POST-HARVEST PHYSIOLOGY AND MOLECULAR BIOLOGY OF FRUITS VEGETABLES AND FLOWERS: SIGNIFICANT ACHIEVEMENTS AND '

FUTURE PROSPECTS Vijay Paul, Ajay Arora, V. P. Singh and G. C. Srivastava

Division of Plant Physiology Indian Agricultural Research Institute, New Delhi 110 012

The available climatic diversity in India, which includes climates such as; tropical, sub-tropical and temperate, allows us to grow wide range of fruits, vegetables and flowers . Post-harvest losses of fruits, flowers and vegetables are enormous in developing countries due to poor storage and transport facilities. It is estimated that post-harvest loss in India is up to 40 %. Thus, there is need to understand in depth the ripening and senescence process so that these processes can be regulated to save the fruits, vegetables and flowers from post­harvest losses. Fruit ripening and flower senescence are associated with a series of physiological and biochemical changes. These include an increase in hydrolytic enzymes, degradation of macromolecules, increased respiratory activity and a loss of cellular compartmentalization. During ripening/senescence endogenous signals up regulate certain genes whose products show high homology to enzymes known to induce climacteric shift, aroma and flavour besides causing the degradation of proteins, RNA, lipids and chlorophyll etc.

I. Post-harvest physiology and molecular biology of fruits and vegetables

More than a hundred fruit species are grown throughout the world. They serve as food, functional food and nutraceuticals as they are source of energy, vitamins, minerals, dietary fibers, antioxidants and other phytochemicals. There has been a significant change in food consumption habits in the last one decade. Demand for high quality, fresh, nutritious and conveniently prepared food items has increased drastically. The tenet "Let food be thy medicine and medicine be thy food" as said by Hippocrates nearly 2,500 years ago is

-receiving renewed interest. Both , quan titative and qualitative losses occur in fruits between harvest and consumption. As per an estimate about one­third of all the fruits produced worldwide are never consumed by humans. It is therefore desirable to minimize these losses by extensive understanding of processes along with the elucidation of regulatory steps and mechanisms involved during post-harvest period. This is urgently needed not only in view of providing the nutritional security to human beings but also towards the sustained economic gai to the farmers .

Harvest commodities are still livi ng organs. They continue to respire and loose water as if they were still attached to the parental plant, the only difference being that losses are not replaced in the post-harvest environment. They therefore suffer detrimental changes after harvest. These changes include the utilization of energy reserves through respiration, changes in biochemical composition, textural changes, water loss, and increased ethylene production. As a result, both, qualitative and quantitative losses occur in fruits betwe en-harvest and consumptions. Approaches like, controlled atmosphere, modified atmosphere and modified atmosphere package can regulate the on going metabolic processes, respiration and transpiration and so they have become the established practices for extending the post-harvest life of fruits. At present, India is the second largest producer of fruits, and vegetables after China. Our country is producing enough fruits and vegetables but due to two basic problems i.e., insufficient infra-structure and inadequate post-harvest management the estimated average post-harvest losses are quite high. Further, general difference between developed

and developing countries is that more of the losses occur between production and retail sites in developing than in developed countries. Large annual losses due to spoilage thereby makes a meaning to control the ripening, especially in climacteric fruits like; tomato and mango, via some easy and cost-effective approaches suitable for the developing countries in the sub-tropical and tropical regions of the world.

Significant achievements in post-harvest physiology of fruits: Based on the work carried out in the area of post-harvest physiology of fruits/ vegetables in the Division of Plant Physiology the significant findings are being summarized below :­

A. Effect of physical treatments on ripening (i),Gamma radiation: Irradiation dose of 0.12 and 0.15 K Gy helped in maintaining the green colour of the mangoes in variety Amrapali [harvested at green mature (water sinked stage) and stored at 30.0 °Cand RH of 56.0 % up to 15 days] at least for 7 to 10 days. But, this extended duration of green colour for the treated fruits of Amrapali appeared to be of little practical significance because the ripening process was not delayed. Infect, irradiation dose of 0.15 K Gy promoted the ripening process as it was revealed by the visual colour and the softness of the pulp tissue . Higher dose of gamma radiation (0.25 K Gy) when given to green mature fruits of variety Dushehari (stored at 35±3 DC and RH 69±1O %) maintained the green colour. But, again blackening of the skin colour was noticed in treated fruits in comparison with control (Divisional Annual Report, 2002-2000. Pp. 22-24).

(li), UV-C radiation: Exposure ofUV-C light (254 om) at lower doses was reported to trigger the antioxidative system along with plant defense mechanisms. Our results on tomato fruits showed that the treated green mature fruits recorded visual change in the pigmentation but with out any positive effect either on respiration rate or on the ripening rate during the course of ripening. This alteration in the colour/pigmentation of tomato fruits especially with increase in the duration of exposure of UV-C light (254 nm) at lower doses

may be of practical relevance as pigments, especially carotenoids, have nutritional quality with manifold health benefits due to their strong antioxidative property (Divisional Annual Report, 2005-2006. Pp. 20-22).

(iii). Hypoxia: Hypoxia duration of 4 and more than 4 days delayed the ripening of green mature tomato fruits significantly. Even under the hypoxia condition, differential ripening behaviour of two varieties [i.e., Pusa Ruby (fast ripening) and Pusa Gaurav (slow ripening)] was maintained. But, as the duration of hypoxia treatment increased the rottage % also increased (Divisional Annual Report, 2006-2007. Pp. 36-37; Paul and Srivastava, 2006).

(iv). Hypobaric storage: Mango fruits of variety Amrapali when stored in vacuum condition (lower partial pressure) showed delayed ripening by 10­12 days at 25 to 27°C. There was less degradation of chlorophyll and release of ethylene. However the taste of fruits was not normal. When fruits were kept at lower partial pressure along with their storage at lower temperature (12°C) they were fresh, hard and green even up to one month (Divisional Annual Report, 1997-1998. Pp. 22-23; Prasad et al., 1999) .

B. Effect of chemicaIlhormonal treatments on ripening

(i) I-Methylcyclopropene (l-MCP): I-MCP treatment delayed ripening in tomato fruits for varying days depending on concentration, exposure duration and storage temperature. MCP at 5 ull' for 4 h was found to be the most effective dose, which delayed ripening by 4 days in variety Pusa Ruby when fruits were treated at green mature stage. The delay in the respiratory peak was observed due to I-MCP. Treatment of MCPaiso had pounced effect on lycopene accumulation as it was found to be suppressed in treated fruits. Further evaluation of this non-reversible competitive inhibitor of ethylene action on the tomato fruits indicated that the exposure with 0.3 ppm for 24 h was the most effective in delaying the ripening. By this single treatment, reduction in ripening index (%) by 39 and 37 % and red ripe tomatoes (%) by

44 45

POST-HARVEST PHYSIOLOGY AND MOLECULAR BIOLOGY OF FRUITS VEGETABLES AND FLOWERS: SIGNIFICANT ACHIEVEMENTS AND '

FUTURE PROSPECTS Vijay Paul, Ajay Arora, V. P. Singh and G. C. Srivastava

Division of Plant Physiology Indian Agricultural Research Institute, New Delhi 110 012

The available climatic diversity in India, which includes climates such as; tropical, sub-tropical and temperate, allows us to grow wide range of fruits, vegetables and flowers . Post-harvest losses of fruits, flowers and vegetables are enormous in developing countries due to poor storage and transport facilities. It is estimated that post-harvest loss in India is up to 40 %. Thus, there is need to understand in depth the ripening and senescence process so that these processes can be regulated to save the fruits, vegetables and flowers from post­harvest losses. Fruit ripening and flower senescence are associated with a series of physiological and biochemical changes. These include an increase in hydrolytic enzymes, degradation of macromolecules, increased respiratory activity and a loss of cellular compartmentalization. During ripening/senescence endogenous signals up regulate certain genes whose products show high homology to enzymes known to induce climacteric shift, aroma and flavour besides causing the degradation of proteins, RNA, lipids and chlorophyll etc.

I. Post-harvest physiology and molecular biology of fruits and vegetables

More than a hundred fruit species are grown throughout the world. They serve as food, functional food and nutraceuticals as they are source of energy, vitamins, minerals, dietary fibers, antioxidants and other phytochemicals. There has been a significant change in food consumption habits in the last one decade. Demand for high quality, fresh, nutritious and conveniently prepared food items has increased drastically. The tenet "Let food be thy medicine and medicine be thy food" as said by Hippocrates nearly 2,500 years ago is

-receiving renewed interest. Both , quan titative and qualitative losses occur in fruits between harvest and consumption. As per an estimate about one­third of all the fruits produced worldwide are never consumed by humans. It is therefore desirable to minimize these losses by extensive understanding of processes along with the elucidation of regulatory steps and mechanisms involved during post-harvest period. This is urgently needed not only in view of providing the nutritional security to human beings but also towards the sustained economic gai to the farmers .

Harvest commodities are still livi ng organs. They continue to respire and loose water as if they were still attached to the parental plant, the only difference being that losses are not replaced in the post-harvest environment. They therefore suffer detrimental changes after harvest. These changes include the utilization of energy reserves through respiration, changes in biochemical composition, textural changes, water loss, and increased ethylene production. As a result, both, qualitative and quantitative losses occur in fruits betwe en-harvest and consumptions. Approaches like, controlled atmosphere, modified atmosphere and modified atmosphere package can regulate the on going metabolic processes, respiration and transpiration and so they have become the established practices for extending the post-harvest life of fruits. At present, India is the second largest producer of fruits, and vegetables after China. Our country is producing enough fruits and vegetables but due to two basic problems i.e., insufficient infra-structure and inadequate post-harvest management the estimated average post-harvest losses are quite high. Further, general difference between developed

and developing countries is that more of the losses occur between production and retail sites in developing than in developed countries. Large annual losses due to spoilage thereby makes a meaning to control the ripening, especially in climacteric fruits like; tomato and mango, via some easy and cost-effective approaches suitable for the developing countries in the sub-tropical and tropical regions of the world.

Significant achievements in post-harvest physiology of fruits: Based on the work carried out in the area of post-harvest physiology of fruits/ vegetables in the Division of Plant Physiology the significant findings are being summarized below :­

A. Effect of physical treatments on ripening (i),Gamma radiation: Irradiation dose of 0.12 and 0.15 K Gy helped in maintaining the green colour of the mangoes in variety Amrapali [harvested at green mature (water sinked stage) and stored at 30.0 °Cand RH of 56.0 % up to 15 days] at least for 7 to 10 days. But, this extended duration of green colour for the treated fruits of Amrapali appeared to be of little practical significance because the ripening process was not delayed. Infect, irradiation dose of 0.15 K Gy promoted the ripening process as it was revealed by the visual colour and the softness of the pulp tissue . Higher dose of gamma radiation (0.25 K Gy) when given to green mature fruits of variety Dushehari (stored at 35±3 DC and RH 69±1O %) maintained the green colour. But, again blackening of the skin colour was noticed in treated fruits in comparison with control (Divisional Annual Report, 2002-2000. Pp. 22-24).

(li), UV-C radiation: Exposure ofUV-C light (254 om) at lower doses was reported to trigger the antioxidative system along with plant defense mechanisms. Our results on tomato fruits showed that the treated green mature fruits recorded visual change in the pigmentation but with out any positive effect either on respiration rate or on the ripening rate during the course of ripening. This alteration in the colour/pigmentation of tomato fruits especially with increase in the duration of exposure of UV-C light (254 nm) at lower doses

may be of practical relevance as pigments, especially carotenoids, have nutritional quality with manifold health benefits due to their strong antioxidative property (Divisional Annual Report, 2005-2006. Pp. 20-22).

(iii). Hypoxia: Hypoxia duration of 4 and more than 4 days delayed the ripening of green mature tomato fruits significantly. Even under the hypoxia condition, differential ripening behaviour of two varieties [i.e., Pusa Ruby (fast ripening) and Pusa Gaurav (slow ripening)] was maintained. But, as the duration of hypoxia treatment increased the rottage % also increased (Divisional Annual Report, 2006-2007. Pp. 36-37; Paul and Srivastava, 2006).

(iv). Hypobaric storage: Mango fruits of variety Amrapali when stored in vacuum condition (lower partial pressure) showed delayed ripening by 10­12 days at 25 to 27°C. There was less degradation of chlorophyll and release of ethylene. However the taste of fruits was not normal. When fruits were kept at lower partial pressure along with their storage at lower temperature (12°C) they were fresh, hard and green even up to one month (Divisional Annual Report, 1997-1998. Pp. 22-23; Prasad et al., 1999) .

B. Effect of chemicaIlhormonal treatments on ripening

(i) I-Methylcyclopropene (l-MCP): I-MCP treatment delayed ripening in tomato fruits for varying days depending on concentration, exposure duration and storage temperature. MCP at 5 ull' for 4 h was found to be the most effective dose, which delayed ripening by 4 days in variety Pusa Ruby when fruits were treated at green mature stage. The delay in the respiratory peak was observed due to I-MCP. Treatment of MCPaiso had pounced effect on lycopene accumulation as it was found to be suppressed in treated fruits. Further evaluation of this non-reversible competitive inhibitor of ethylene action on the tomato fruits indicated that the exposure with 0.3 ppm for 24 h was the most effective in delaying the ripening. By this single treatment, reduction in ripening index (%) by 39 and 37 % and red ripe tomatoes (%) by

44 45

100 and 85 % were recorded in Pusa Ruby after 7 and 14 days after harvest respectively. I-MCP delayed the ripening by 5-7 days and 8-10 days in tomato fruits of Pusa Ruby and Pusa Gaurav respectively even if the fruits were stored at higher temperature range of29 to 32°C and RH of37.0±10 %. Effectiveness of I-MCPtreatment (2 and 4 ppm for 4 hours) in delaying the process of ripening at storage temperature of 30°C was also demonstrated at breaker and turning stages for both, thin and thick-skinned tomato fruits. Data on physiological

J loss in weight (PLW) in tomato fruits remained unaffected by I-MCP treatment. I-MCP treatment on mango fruits, on the other hand, showed no beneficial effect at least for the doses and the conditions, which were used by us (Divisional Annual Report, 2002-2003. Pp. 16-19; 2003-2004. Pp.21;2005-2006.Pp.20-22;2006-2007.Pp.35; Pushpalatha et al., 2006).

(ii). Ethanol: Ethanol vapours were found to be effective in delaying the ripening of tomato fruits. Delay in ripening of tomato fruits for different durations ranging from 10-32 days was noticed depending upon the concentration of ethanol, duration of its exposure and stage at which fruits were treated. Most effective treatment was 8 and 10 ml/kg of fruit for 24 h, which caused delay in ripening up to 30 days for green mature tomato fruit when stored at 25°C. For both the varieties, lower doses could not delay the ripening substantially, while higher doses caused some adverse effects. Ethanol dose of 8 ml/kg and beyond is causing scar tissue damage for Pusa Gaurav. While 16-ml/kg and beyond caused similar affect on Pusa Ruby. Optimum dose of ethanol required to delay the ripening without any adverse effect was therefore found to be the variety dependent. Ethanol mediated delay in the ripening of tomato f~uit was found to be associated with the better membrane stability of pericarp tissue of tomato fruit. Biochemical analysis showed delay in climacteric rise in respiration and ethylene peaks in ethanol treated fruits. Activity of PG and PME enzymes were very low in treated fruits compared to untreated fruits (Zeng et al., 1996;

Sharma-Natu et al., 2003; Srivastava and Paul, 2003; Divisional Annual Report, 1997­1998. Pp.23-24; 2005-2006. Pp. 20-22; 2006-2007. Pp.35).

(iii): Hormonal regulation of fruit ripening: BA (10 ppm) effectively delayed both respiratory burst and ethylene peak in banana fruit when harvested and treated at green mature stage. While, ABA (100 ppm) advanced the ethylene production and rate of reapiration. Biochemical studies indicated slower chlorophyll degradation, prolonged activities of SOD, catalase and peroxidase and less ion leakage in BA treated fruits (Divisional Annual Report, 2000-2001. Pp. 17-20).

C. Membrane stability of tomato pericarp tissue and its ripening behaviour: Electrolyte efflux (%) pattern for attached and detached tomato fruits in slow (Pusa Gaurav) and fast (Pusa Ruby) ripening varieties indicated that slow ripening was not characterized by better membrane stability. Electrolyte efflux (%) was found to be more related with ripening of tomato fruits on plant rather than for fruits harvested at green mature stage and stored. Rate of electrolyte efflux was not influenced by slow or fast ripening behaviour of tomato varieties. It was concluded that the electrolyte efflux (%) could not be a suitable criterion to assess the slow and fast ripening behaviour of tomato varieties either for detached or attached fruits. This study also ruled out the slow and fast ripening behaviour of varieties in view of differences in the quantity of hypothetical "ripening inhibitor substance" being translocated to the fruit undergoing ripening while attached to the plant (Divisional Annual Report , 2004-2005. Pp. 21; Paul, V. et al., 2005).

D. Anatomy of climacteric fruits and their ripening behaviour (l). Mango fruit: Basic anatomical differences among different and contrasting varieties (with respect to ripening behaviour) were noticed in terms of surface anatomy/morphological features such as; thickness of cuticle, number of hypodermal layers, thickness of hypodermis, compactness of hypodermal cells, intensity of staining of exocarp

region with safranin, cell density, cell size and extent of intercellular spaces. In general, thickness of cuticle was less (8 .2 urn) for Langra in comparison with other three varieties of mango where the thickness was almost comparable i. e., 11.4, 13.7 and 11.8 urn. Thickness data for epicarp region indicated more prominent epicarp in Langra (70 urn) and Ramkela (68 urn) and Amrapali (65 urn) in comparison with Dushehari (53 urn), The observation therefore provided the anatomical basis of tough and thicker skin for Langra, Rarnkela and Amrapali in contrast with Dushehari. Morphometric data along with visual observations in terms of I. Apparent cell density, 2. Apparent cell size and 3. Extent of intercellular space revealed that the higher cell density in Langra followed by Amrapali, Dushehari and Rarnkela for epicarp and for outer mesocarp regions were due to the smaller cell size and less of available intercellular spaces for variety Langra followed by Arnrapali, Dushehari and Ramkela. The obtained results therefore indicated higher surface to the volume ratio for Langra followed by Amrapali, Dushehari and Ramkela. Since, the rate of ripening in these varieties also followed the same order i. e., Langra followed by Amrapali, Dushehari and Rarnkela so a possible correlation could exist between the rate of ripening in varieties of mango and anatomical features in epicarp and mesocarp regions of the fruits. There is a possibility that cell size/cell density might be regulating the rate of ripening via determining the basal metabolic rate. Work is in progress in this area (Divisional Annual Report, 2003-2004. Pp. 21-24; 2004-2005. Pp. 21-22; 2005-2006. Pp. 23-24; Paul, V. et al., 2007. In press).

(ii), Tomato fruit: Tendency of conversion of hair base cell of trichome into the lenticel was less for Pusa Gaurav in comparison with Pusa Ruby and therefore number of lenticels was comparatively less in Pusa Gaurav. At different stages of ripening as well as at different days after harvest significantly lower values for the sites of gaseous exchange/ water loss (lenticels along with trichomes) were recorded in Pusa Gaurav than the Pusa Ruby. The

results thus indicated the varietal difference in surface morphologicaVanatomical features of fruit and this might be contributing for differences in rate of respiration and PLW % (Divisional Annual Report, 2006-2007. Pp. 37-38; Paul and Srivastava, 2006).

E. Physiological basis for the varietal variation in the ripening behaviour of climacteric fruits (i). Clonal variation towards ripening in mango fruits: Amrapali and Dashehari clones .produced measurable levels of ethylene but did not show climacteric pattern. Two ethylene peaks were recorded during ripening; one at initial stage and the other at final stage of ripening. NADP-malic enzyme activity and respiratory climacteric followed a parallel increase. ACC-synthase was not found limited, whereas ethylene forming enzyme (EFE) was limited and resulted in the accumulation ofACC with the progress in ripening in both the cultivars of mango. Embryo (stones) also produced considerable amount of ethylene (Divisional Annual Report, 1997-1998. Pp. 21-22; Zeng et al., 1995; Zeng et al., 1996; Srivastava et al., 1996; Pandey et al., 1998; Reddy and Srivastava, 1998; Reddy and Srivastava, 1999a; Reddy and Srivastava, 1999b; Prasad et al., 1999; Reddy and Srivastava, 2001; Reddy and Srivastava, 2003; Komal Mathur and Srivastava, 2005).

(ii). Respiration rate for attached and detached fruits of tomato: Tomato fruits of Pus a Gaurav (a slow ripening variety) when harvested at green mature stage and stored or when harvested directly from the plant at different ripening stages exhibited significantly lower rate of respiration in comparison with variety Pusa Ruby (a fast ripening variety). Pusa Gaurav also showed lower PLW % throughout the storage duration. In tomato fruits, anatomical features (density of trichome base cells and lenticels) and surface morphology itself (dimension of stem scar region) appear to regulate the ripening rate and ripening-associated changes possibly by determining the 0 2 to CO ratio in the

2 microenvironment of the tomato fruit. The results thus indicated the varietal difference in surface morphologicaVanatomical features of fruit and this

46 47

100 and 85 % were recorded in Pusa Ruby after 7 and 14 days after harvest respectively. I-MCP delayed the ripening by 5-7 days and 8-10 days in tomato fruits of Pusa Ruby and Pusa Gaurav respectively even if the fruits were stored at higher temperature range of29 to 32°C and RH of37.0±10 %. Effectiveness of I-MCPtreatment (2 and 4 ppm for 4 hours) in delaying the process of ripening at storage temperature of 30°C was also demonstrated at breaker and turning stages for both, thin and thick-skinned tomato fruits. Data on physiological

J loss in weight (PLW) in tomato fruits remained unaffected by I-MCP treatment. I-MCP treatment on mango fruits, on the other hand, showed no beneficial effect at least for the doses and the conditions, which were used by us (Divisional Annual Report, 2002-2003. Pp. 16-19; 2003-2004. Pp.21;2005-2006.Pp.20-22;2006-2007.Pp.35; Pushpalatha et al., 2006).

(ii). Ethanol: Ethanol vapours were found to be effective in delaying the ripening of tomato fruits. Delay in ripening of tomato fruits for different durations ranging from 10-32 days was noticed depending upon the concentration of ethanol, duration of its exposure and stage at which fruits were treated. Most effective treatment was 8 and 10 ml/kg of fruit for 24 h, which caused delay in ripening up to 30 days for green mature tomato fruit when stored at 25°C. For both the varieties, lower doses could not delay the ripening substantially, while higher doses caused some adverse effects. Ethanol dose of 8 ml/kg and beyond is causing scar tissue damage for Pusa Gaurav. While 16-ml/kg and beyond caused similar affect on Pusa Ruby. Optimum dose of ethanol required to delay the ripening without any adverse effect was therefore found to be the variety dependent. Ethanol mediated delay in the ripening of tomato f~uit was found to be associated with the better membrane stability of pericarp tissue of tomato fruit. Biochemical analysis showed delay in climacteric rise in respiration and ethylene peaks in ethanol treated fruits. Activity of PG and PME enzymes were very low in treated fruits compared to untreated fruits (Zeng et al., 1996;

Sharma-Natu et al., 2003; Srivastava and Paul, 2003; Divisional Annual Report, 1997­1998. Pp.23-24; 2005-2006. Pp. 20-22; 2006-2007. Pp.35).

(iii): Hormonal regulation of fruit ripening: BA (10 ppm) effectively delayed both respiratory burst and ethylene peak in banana fruit when harvested and treated at green mature stage. While, ABA (100 ppm) advanced the ethylene production and rate of reapiration. Biochemical studies indicated slower chlorophyll degradation, prolonged activities of SOD, catalase and peroxidase and less ion leakage in BA treated fruits (Divisional Annual Report, 2000-2001. Pp. 17-20).

C. Membrane stability of tomato pericarp tissue and its ripening behaviour: Electrolyte efflux (%) pattern for attached and detached tomato fruits in slow (Pusa Gaurav) and fast (Pusa Ruby) ripening varieties indicated that slow ripening was not characterized by better membrane stability. Electrolyte efflux (%) was found to be more related with ripening of tomato fruits on plant rather than for fruits harvested at green mature stage and stored. Rate of electrolyte efflux was not influenced by slow or fast ripening behaviour of tomato varieties. It was concluded that the electrolyte efflux (%) could not be a suitable criterion to assess the slow and fast ripening behaviour of tomato varieties either for detached or attached fruits. This study also ruled out the slow and fast ripening behaviour of varieties in view of differences in the quantity of hypothetical "ripening inhibitor substance" being translocated to the fruit undergoing ripening while attached to the plant (Divisional Annual Report , 2004-2005. Pp. 21; Paul, V. et al., 2005).

D. Anatomy of climacteric fruits and their ripening behaviour (l). Mango fruit: Basic anatomical differences among different and contrasting varieties (with respect to ripening behaviour) were noticed in terms of surface anatomy/morphological features such as; thickness of cuticle, number of hypodermal layers, thickness of hypodermis, compactness of hypodermal cells, intensity of staining of exocarp

region with safranin, cell density, cell size and extent of intercellular spaces. In general, thickness of cuticle was less (8 .2 urn) for Langra in comparison with other three varieties of mango where the thickness was almost comparable i. e., 11.4, 13.7 and 11.8 urn. Thickness data for epicarp region indicated more prominent epicarp in Langra (70 urn) and Ramkela (68 urn) and Amrapali (65 urn) in comparison with Dushehari (53 urn), The observation therefore provided the anatomical basis of tough and thicker skin for Langra, Rarnkela and Amrapali in contrast with Dushehari. Morphometric data along with visual observations in terms of I. Apparent cell density, 2. Apparent cell size and 3. Extent of intercellular space revealed that the higher cell density in Langra followed by Amrapali, Dushehari and Rarnkela for epicarp and for outer mesocarp regions were due to the smaller cell size and less of available intercellular spaces for variety Langra followed by Arnrapali, Dushehari and Ramkela. The obtained results therefore indicated higher surface to the volume ratio for Langra followed by Amrapali, Dushehari and Ramkela. Since, the rate of ripening in these varieties also followed the same order i. e., Langra followed by Amrapali, Dushehari and Rarnkela so a possible correlation could exist between the rate of ripening in varieties of mango and anatomical features in epicarp and mesocarp regions of the fruits. There is a possibility that cell size/cell density might be regulating the rate of ripening via determining the basal metabolic rate. Work is in progress in this area (Divisional Annual Report, 2003-2004. Pp. 21-24; 2004-2005. Pp. 21-22; 2005-2006. Pp. 23-24; Paul, V. et al., 2007. In press).

(ii), Tomato fruit: Tendency of conversion of hair base cell of trichome into the lenticel was less for Pusa Gaurav in comparison with Pusa Ruby and therefore number of lenticels was comparatively less in Pusa Gaurav. At different stages of ripening as well as at different days after harvest significantly lower values for the sites of gaseous exchange/ water loss (lenticels along with trichomes) were recorded in Pusa Gaurav than the Pusa Ruby. The

results thus indicated the varietal difference in surface morphologicaVanatomical features of fruit and this might be contributing for differences in rate of respiration and PLW % (Divisional Annual Report, 2006-2007. Pp. 37-38; Paul and Srivastava, 2006).

E. Physiological basis for the varietal variation in the ripening behaviour of climacteric fruits (i). Clonal variation towards ripening in mango fruits: Amrapali and Dashehari clones .produced measurable levels of ethylene but did not show climacteric pattern. Two ethylene peaks were recorded during ripening; one at initial stage and the other at final stage of ripening. NADP-malic enzyme activity and respiratory climacteric followed a parallel increase. ACC-synthase was not found limited, whereas ethylene forming enzyme (EFE) was limited and resulted in the accumulation ofACC with the progress in ripening in both the cultivars of mango. Embryo (stones) also produced considerable amount of ethylene (Divisional Annual Report, 1997-1998. Pp. 21-22; Zeng et al., 1995; Zeng et al., 1996; Srivastava et al., 1996; Pandey et al., 1998; Reddy and Srivastava, 1998; Reddy and Srivastava, 1999a; Reddy and Srivastava, 1999b; Prasad et al., 1999; Reddy and Srivastava, 2001; Reddy and Srivastava, 2003; Komal Mathur and Srivastava, 2005).

(ii). Respiration rate for attached and detached fruits of tomato: Tomato fruits of Pus a Gaurav (a slow ripening variety) when harvested at green mature stage and stored or when harvested directly from the plant at different ripening stages exhibited significantly lower rate of respiration in comparison with variety Pusa Ruby (a fast ripening variety). Pusa Gaurav also showed lower PLW % throughout the storage duration. In tomato fruits, anatomical features (density of trichome base cells and lenticels) and surface morphology itself (dimension of stem scar region) appear to regulate the ripening rate and ripening-associated changes possibly by determining the 0 2 to CO ratio in the

2 microenvironment of the tomato fruit. The results thus indicated the varietal difference in surface morphologicaVanatomical features of fruit and this

46 47

might be contributing for differences in. rate. of respiration and PLW % and so in the nperung behaviour. This also provides one of the reasons for the varietal variability in ripening behaviour of the tomato fruits (Divisional Annual Report, 2005-2006. Pp . 22-23 ; Paul and Srivastava, 2006).

(iii). Role of stem scar tissue a~d surface morphology of tomato fruits: Blocking t~e stem scar tissue of the tomato fruit showed drastic eff~ct

in lowering the rate of respiration of the fruits, Blocking of stem scar region also diminishe~ or completely inhibits the climacteric rise in vanety Pusa Ruby. Fruits of Pusa Gaurav sho~ed

significantly lower surface area of stem scar region and also lower rate of respiration, lower physiological loss in weight along with slower

• ripening in comparison to Pusa Ruby. More resistance towards gaseous exchange and thereby lower ° to CO ratio inside the fruits of Pusa Gaurav i~ contra~t to Pusa Ruby could possibly be one of the reasons responsible for the obtained varietal difference in ripening behaviour of the tomato fruits. Effect of blocking the stem scar region was also found to be dependent ~n the maturity stage of the fruits itself. Maximum reduction in ripening and respiration was recorded for fruits blocked at green mature stage. This suppressive effect was found to be fa~ed away gradually with subsequent higher mat~nt~. stages i.e., breaker and turning. The results sIgmf~ed t~e

role for the area of the stem scar region III

determining not only the rate of ripening but also in the respiratory and climacteric rise as well. The study clearly pointed out the role, importance a~d

significance of internal microenvironment and Its alternation on ripening and ripening related changes along with providing one of the. r~aso~s for explaining the existing varietal variations In t?e ripening and respiratory behaviour of tomato fruits (Paul and Srivastava, 2006).

F. Enhancement of nutritional quality of fruits by manipulating carotenoid biosynthe.tic pathway genes: Only next to food, secunty,

. nutritional security is an important national goal.

In India, 63 % children (under 5) are malnourished, highest in the world excluding only Ne~al and Bangladesh and 33 % infants are borne ~Ith low birth weight. This is due to inadequate I~take of essential nutrients like nutritious proteins and vitamins. According to a survey conducted by National Nutrition Monitoring Bureau (NNMB), major nutritional deficiency among chil~ren ,is protein energy malnutrition (PEM) and vitamin

deficiency. The NNMB data (I :88-:0) r~v~aled

that there is a higher degree of vitarrun deficiency than PEM. Therefore, we are interested in a long­term goal of making available convenient and cost­effective methods of vitamin fortification through transgenic fruits and vegetables, including specific antioxidants.

In photosynthetic tissues, carotenoi~s are synthesized within chloroplasts and function as photoprotectants. In non-green plant tissues (su~h

as banana fruit), carotenoids accumulate III

chromoplasts. A significant increase in .tot~l

carotenoids occurs during fruit ripening, which IS primarily due to the accumulation of tw~ car?tenes. The biosynthesis of these carotenoids IS be~t

understood in tomato. Phytoene, the first carotenoid in the pathway, is synthesized from two molecules of geranyl geranyl diphosphate by phytoe~e

synthase (PSY). Subsequently, phytoene ~s

converted to Lycopene and beta-carotene. In this pathway, the first step catalyzed by phytoe~e

synthase is rate limiting and the level ofPSY protem is the main factor determining lycopene and beta­carotene production in ripening fruits. Thus, we ultimately aim to introgress the phytoene synthase gene encoding PSY protein, a~d othe~ related ge~es,

which are specific to ripemng fruits from high carotenoid containing species to cultivated elite low carotenoid containing cultivars to increase various kinds of carotenoids in fruits.

It has been shown that tomato fruits contain high levels of beta-carotene at the expense of lycopene. It is also known that phytoene synthase encoded, by PsyJ gene is the pacemaker en~yme .in carotenoid biosynthesis in ripening fruit and ItS expression is regulated at the level of transcription.

48

Thus, it is envisaged that if the PsyJ gene will over­express in ripening fruits, both lycopene as well as beta-carotene levels would increase substantially. Earlier, it has been demonstrated that PsyJ gene product catalyses the rate-limiting step in the carotenoid pathway of ripening tomato fruits, where it was observed that inhibition of Psyl gene expression by antisense RNA expression, led to the absence of lycopene synthesis in antisense ripe fruits

Genetic stocks are available for use in analyzing genetic variation in carotenoid biosynthesis and accumulation. It is thus possible to assess the relationship between variations in carotenoid content, particularly beta-carotene, and expression of the carotenoid biosynthesis pathway genes, which in tum will help identify key genes regulating carotenoid accumulation. Thus, we would like to study expression analysis of carotenoid biosynthesis pathway genes in banana by utilizing mutants and wild species by cloning.

(Divisional Annual Report, 2005-2006. Pp. 24-25)

II. Post-harvest physiology and molecular biology of flowers

Flowers are highly perishable in nature and it is estimated that nearly 30 % are rendered unmarketable due to post-harvest quality loss. This loss is mainly because of senescing flowers and leaves of cut flowers .

There are certain flowers like rose and carnation where floral senescence is regulated in part by ethylene production. However, there is another big group of plants like daylily, sandersonia, tulip, narcissus, irish, hollandica, gladiolus etc . whose flowers are insensitive to ethylene. If we increase the physiological level of this hormone the senescence is not hastened in these flowers or if we inhibit its production in flower or its action then senescence can not be delayed. Hence efforts are being made to know how senescence is triggered and proceeds in such ethylene insensitive flowers.

Significant achievements in post-harvest physiology of flowers: Based on the work carried out in the area of post-harvest physiology of fruits/

49

vegetables in the Division of Plant Physiology the significant findings are being summarized below:­

A. Extension of vase life of rose flower using ethanol and sucrose: Presence of 3 % sucrose in the vase solution (water) enhanced the vase life of flowers in "First Red" variety of rose by two days. Application of3 % ethanol also had a positive effect on the vase life. However, a combination of 3 % ethanol and 3 % sucrose gave the best results. An enhancement of 7 days was observed in the vase life of flowers . In the treated flowers, water potential was higher, ABA accumulation was lower and ethylene production was reduced (Divisional Annual Report, 2005-2006. Pp. 24-28; 2006-2007. In press; Kumar et al., 2007).

B. Senescence in chrysanthemum flower: In vase solution, flowers treated with BA and sucrose showed lesser leakage of ion. Activity of PG and PME was found to be less in flowers kept in sucrose or BA solution. In all the tested variety, flowers in BA or sucrose showed higher protein content. On the other hand flowers kept in ABA or control showed greater decline in protein content. Reverse was the trend for protease activity. Further work on antioxidative enzymes and H 0 also

2 2production supported the BA and sucrose mediated delay in senescence of flowers (Divisional Annual Report, 1998-1999. Pp. 21-24; Elanchezhian, and .~ Srivastava, 2001a; 2001b).

C. Alpha lipoic acid regulated flower senescence in gladiolus: Alpha lipoic acid or thiotctic acid (1, 2-ditholane 3-pentanoic acid) is a (thiol) disulphide compound found naturally in mitochondria. This is a coenzyme and is essential for the activity of enzymes such as pyruvate dehydrogenase, alpha ketoglutarate dehydrogenase and glycine decarboxylase. It is also involved in the glycolysis process. There are various reports in animals and human being that alpha lipoic acid (LA) supplementation increase antioxidant status and ameliorate oxidative stress. There is no report so far on the role of alpha LA in the regulation senescence in plants; hence this work was taken up. Application of 100 ppm LA in vase solution along with sucrose solution increased vase life

might be contributing for differences in. rate. of respiration and PLW % and so in the nperung behaviour. This also provides one of the reasons for the varietal variability in ripening behaviour of the tomato fruits (Divisional Annual Report, 2005-2006. Pp . 22-23 ; Paul and Srivastava, 2006).

(iii). Role of stem scar tissue a~d surface morphology of tomato fruits: Blocking t~e stem scar tissue of the tomato fruit showed drastic eff~ct

in lowering the rate of respiration of the fruits, Blocking of stem scar region also diminishe~ or completely inhibits the climacteric rise in vanety Pusa Ruby. Fruits of Pusa Gaurav sho~ed

significantly lower surface area of stem scar region and also lower rate of respiration, lower physiological loss in weight along with slower

• ripening in comparison to Pusa Ruby. More resistance towards gaseous exchange and thereby lower ° to CO ratio inside the fruits of Pusa Gaurav i~ contra~t to Pusa Ruby could possibly be one of the reasons responsible for the obtained varietal difference in ripening behaviour of the tomato fruits. Effect of blocking the stem scar region was also found to be dependent ~n the maturity stage of the fruits itself. Maximum reduction in ripening and respiration was recorded for fruits blocked at green mature stage. This suppressive effect was found to be fa~ed away gradually with subsequent higher mat~nt~. stages i.e., breaker and turning. The results sIgmf~ed t~e

role for the area of the stem scar region III

determining not only the rate of ripening but also in the respiratory and climacteric rise as well. The study clearly pointed out the role, importance a~d

significance of internal microenvironment and Its alternation on ripening and ripening related changes along with providing one of the. r~aso~s for explaining the existing varietal variations In t?e ripening and respiratory behaviour of tomato fruits (Paul and Srivastava, 2006).

F. Enhancement of nutritional quality of fruits by manipulating carotenoid biosynthe.tic pathway genes: Only next to food, secunty,

. nutritional security is an important national goal.

In India, 63 % children (under 5) are malnourished, highest in the world excluding only Ne~al and Bangladesh and 33 % infants are borne ~Ith low birth weight. This is due to inadequate I~take of essential nutrients like nutritious proteins and vitamins. According to a survey conducted by National Nutrition Monitoring Bureau (NNMB), major nutritional deficiency among chil~ren ,is protein energy malnutrition (PEM) and vitamin

deficiency. The NNMB data (I :88-:0) r~v~aled

that there is a higher degree of vitarrun deficiency than PEM. Therefore, we are interested in a long­term goal of making available convenient and cost­effective methods of vitamin fortification through transgenic fruits and vegetables, including specific antioxidants.

In photosynthetic tissues, carotenoi~s are synthesized within chloroplasts and function as photoprotectants. In non-green plant tissues (su~h

as banana fruit), carotenoids accumulate III

chromoplasts. A significant increase in .tot~l

carotenoids occurs during fruit ripening, which IS primarily due to the accumulation of tw~ car?tenes. The biosynthesis of these carotenoids IS be~t

understood in tomato. Phytoene, the first carotenoid in the pathway, is synthesized from two molecules of geranyl geranyl diphosphate by phytoe~e

synthase (PSY). Subsequently, phytoene ~s

converted to Lycopene and beta-carotene. In this pathway, the first step catalyzed by phytoe~e

synthase is rate limiting and the level ofPSY protem is the main factor determining lycopene and beta­carotene production in ripening fruits. Thus, we ultimately aim to introgress the phytoene synthase gene encoding PSY protein, a~d othe~ related ge~es,

which are specific to ripemng fruits from high carotenoid containing species to cultivated elite low carotenoid containing cultivars to increase various kinds of carotenoids in fruits.

It has been shown that tomato fruits contain high levels of beta-carotene at the expense of lycopene. It is also known that phytoene synthase encoded, by PsyJ gene is the pacemaker en~yme .in carotenoid biosynthesis in ripening fruit and ItS expression is regulated at the level of transcription.

48

Thus, it is envisaged that if the PsyJ gene will over­express in ripening fruits, both lycopene as well as beta-carotene levels would increase substantially. Earlier, it has been demonstrated that PsyJ gene product catalyses the rate-limiting step in the carotenoid pathway of ripening tomato fruits, where it was observed that inhibition of Psyl gene expression by antisense RNA expression, led to the absence of lycopene synthesis in antisense ripe fruits

Genetic stocks are available for use in analyzing genetic variation in carotenoid biosynthesis and accumulation. It is thus possible to assess the relationship between variations in carotenoid content, particularly beta-carotene, and expression of the carotenoid biosynthesis pathway genes, which in tum will help identify key genes regulating carotenoid accumulation. Thus, we would like to study expression analysis of carotenoid biosynthesis pathway genes in banana by utilizing mutants and wild species by cloning.

(Divisional Annual Report, 2005-2006. Pp. 24-25)

II. Post-harvest physiology and molecular biology of flowers

Flowers are highly perishable in nature and it is estimated that nearly 30 % are rendered unmarketable due to post-harvest quality loss. This loss is mainly because of senescing flowers and leaves of cut flowers .

There are certain flowers like rose and carnation where floral senescence is regulated in part by ethylene production. However, there is another big group of plants like daylily, sandersonia, tulip, narcissus, irish, hollandica, gladiolus etc . whose flowers are insensitive to ethylene. If we increase the physiological level of this hormone the senescence is not hastened in these flowers or if we inhibit its production in flower or its action then senescence can not be delayed. Hence efforts are being made to know how senescence is triggered and proceeds in such ethylene insensitive flowers.

Significant achievements in post-harvest physiology of flowers: Based on the work carried out in the area of post-harvest physiology of fruits/

49

vegetables in the Division of Plant Physiology the significant findings are being summarized below:­

A. Extension of vase life of rose flower using ethanol and sucrose: Presence of 3 % sucrose in the vase solution (water) enhanced the vase life of flowers in "First Red" variety of rose by two days. Application of3 % ethanol also had a positive effect on the vase life. However, a combination of 3 % ethanol and 3 % sucrose gave the best results. An enhancement of 7 days was observed in the vase life of flowers . In the treated flowers, water potential was higher, ABA accumulation was lower and ethylene production was reduced (Divisional Annual Report, 2005-2006. Pp. 24-28; 2006-2007. In press; Kumar et al., 2007).

B. Senescence in chrysanthemum flower: In vase solution, flowers treated with BA and sucrose showed lesser leakage of ion. Activity of PG and PME was found to be less in flowers kept in sucrose or BA solution. In all the tested variety, flowers in BA or sucrose showed higher protein content. On the other hand flowers kept in ABA or control showed greater decline in protein content. Reverse was the trend for protease activity. Further work on antioxidative enzymes and H 0 also

2 2production supported the BA and sucrose mediated delay in senescence of flowers (Divisional Annual Report, 1998-1999. Pp. 21-24; Elanchezhian, and .~ Srivastava, 2001a; 2001b).

C. Alpha lipoic acid regulated flower senescence in gladiolus: Alpha lipoic acid or thiotctic acid (1, 2-ditholane 3-pentanoic acid) is a (thiol) disulphide compound found naturally in mitochondria. This is a coenzyme and is essential for the activity of enzymes such as pyruvate dehydrogenase, alpha ketoglutarate dehydrogenase and glycine decarboxylase. It is also involved in the glycolysis process. There are various reports in animals and human being that alpha lipoic acid (LA) supplementation increase antioxidant status and ameliorate oxidative stress. There is no report so far on the role of alpha LA in the regulation senescence in plants; hence this work was taken up. Application of 100 ppm LA in vase solution along with sucrose solution increased vase life

· tremendously by delaying the senescence.

Initial increase in the fresh weight of spikes in both the vase solution (treated and control) can be attributed to the increased requirement of the spikes for opening of flowers. The decline in fresh weight is due to less water uptake, high respiration rate and also membrane leakage. In the absence of current photoassimilates, reserved carbohydrates are used in respiration. When fresh weight of flower spikes declined below its initial fresh weight then the flower spikes are said to have lost their vase life quality and this is treated as vase life. The vase life was extended significantly with the treatment of lipoic acid.

Membrane stability index (MSI %) represents the ,change in the permeability 'of membrane. Reduction in MSI indicates status of ion leakage or solute leakage. There is a sharp decline in MSI in both the varieties . Membrane deterioration leads to the loss of intracellular compartmentalization, an important feature of senescence. This results in ion and water leakage (cells are not able to retain required ions and water). Membrane stability and fluidity is also lost by the direct attack of free radicals on lipids and protein the constituents of bilayer. MSI was however alleviated by LA treatment.

The thiobarbituric acid reactive substances (TBARS) increased with the senescence indicating increased level of lipid peroxidation. TBARS content was less in LA treated flowers in both the varieties in comparison to control. The LA could delay the senescence by delaying lipid peroxidation (through reduction in TBARS) and maintain higher MSI for the longer duration -Thus LA is beneficial against oxidative lipid damage .

Lipoxygenase are a class of enzymes, which catalyze the hydroperoxidation of polyunsaturated fatty acids, especially in cell membrane. Lipoxygenase activity increases during the flower senescence. Thus, an increase in LOX activity represents an increase in the cell membrane permeability or reduction in MSI. There is a strong temporal correlation during senescence between

50

changes in LOX activity and superoxide radical production by the membranes. From the less LOX activity in LA treated flowers it can be conceived that the free radicals produced through membrane associated LOX may be scavenged by LA. This may contribute to some extent for the extension of vase life of spikes held in LA solution,

Plants have evolved wide range of mechanism to counteract the oxidative stress. Cellular damage caused by lipid peroxidation might be reduced or prevented by protective mechanism involving free radical and peroxide scavenging enzymes such as Superoxide dismutase(SOD), catalase (CAT), glutathione reductase, ascorbate peroxidase etc.

SOD is generally considered key enzyme in the regulation of intracellular concentration of superoxide radical (02.-) and peroxide which can react in Haber-Weiss reaction to form hydroxyl radical leading to peroxidation. Initially activity of SOD is slightly increased then there was a decline at later stage during senescence. SOD activity was higher and retained for longer duration in the flowers treated by LA.

Hydrogen peroxide-CH20) produced during the dismutation of superoxide radicals by SOD is scavenged by enzyme catalase. Activity of catalase is initially increased but declined at latter stages of senescence. Catalase activity was retained at higher level in the flowers treated with LA in comparison to control in both the cultivars. This is because LA directly scavenges free radicals.

Glutathione reductase (GR) catalyses the reduction of glutathione disulphide with the accompanying oxidation of NADPH. GR activity is decreased as the senescence proceeds after initial increase. Reduction in GR activity results in ~

decline in the level of reduced glutathione, known to be an important factor in preventing oxidative injuries. The LA supplemented gladiolus flowe rs showed increased GR activity over control. Hence it can be conceived that LA delayed the senescence by increasing/retaining GR activity for longer period.

Membrane bound ascorbate peroxidase (AP) is found to scavenge the H202which is produced by

the activity of SOD on the superoxide radical. The ascorbate activity was found to be directly correlated with the reduction in free radical induced damage. LA treated flowers showed enhanced level of AP even at later stages of senescence over the control. Thus increased level of AP may also be one of the reason s in postponing the senescence by scavenging H202and preventing membrane damage.

On the basis of this study it is clear that LA can increase or retain the activity of SOD, CAT, GR, and AP thereby increasing antioxidant status of these enzymes to is scavenge free radicals produced during senescence. Apart from this LA can reduce lipid peroxidation by decreasing lipoxygenase activity and TBARS contents which in turn prevent or delay the membrane damage to some extent which ultimately leads to the delay in flower senescence. Thus LA acts as a free radical scavenger and hence delayed the flower senescence in gladiolus flowers .

There was a decline in protein content during senescence. Flower senescence is mainly associated with the loss of protein. The protein content is reduced due to little de novo synthesis and considerable protein degradation It is understood that free radicals attack amino acid residues of proteins causing conformational changes in proteins causing them to be recognized by specific proteases for degradation.Also a change in membrane lipid composition induces conformational change s in proteins , by providing an unfavorable environment deleteriously affecting the activity of key enzymes. (The proteins are excluded into liquid crystalline domain from gel phase liquid domain which progressively increases during senescence. Proteins trapped in the gel phase are degraded by proteases. It is observed that LA treated flowers maintained higher protein over control. It may be because LA acts as a free radical scavenger.

(Arora et al., 2002; Singh and Jegadheesan, 2003; Srivastava, 2003; Arora, 2007a; Arora, 2007b. In press).

51

D. 5-Su lp hosa li cyli c acid delayed the senescence and increased the vase life of gladiolus flowers: Hundred ppm of 5­sulphosalicylic acid (5-SSA) along with 2 % sucrose in holding solution delayed the senescence of gladiolus flowers and consequen tly increased vase life up to 11.2 days while in control it was 5.3 days. The flower quality was improved in terms of percentage flowering , fresh weight and water uptake. Treated flowers also exhibited delayed and reduced ion leakage as well as respiration rate. Superoxide dismutase (SOD) and catalase (CAT) activities were maintained at higher level for longer time in treated flowers to scavenge free radicals. Thiobarbituric acid reactive substances (TBARS) content and lipoxygenase (LOX) activity were also kept in check by 5-SSA thus reducing the level of peroxidation. It is proposed and proved that 5-SSA delayed senescence by acting as a free radical scavenger (Ezhilmathi et al., 2007).

E. Role of polyamines in flower senescence: Polyamines (PA) are now regarded as a new class of growth substances and are well known for their antisenescence effects on animal and plant tissue. The polyamines-spermidine and spermine occur ubiquitously in the plant kingdom, together with their diamine precursor putrescine.

Treatment with either of 100 ppm i.e. spermine, spermidine and putrescine along with 2 % sucrose solution increased the vase life to 9.6 and 8.4 days in Dhanwantari and Snow Princess, respectively by spermine and then followed by spermidine, putrescine and control. Spermine and spermidine could successfully extend vase life of flowers by maintaining higher MSI, protein, higher uptake of vase solution and thus maintaining higher fresh weight for longer duration. Spermine and spermidine were able to reduce the activity of lipoxygenase enzyme and lipid peroxidation that are responsible for membrane deterioration and loss of com partmentalization. Also, during the senescence activi ties of various antioxi dant enzymes viz. SOD , CAT, glutat hione redu ctase (GR) and ascorbate peroxidase (AP) were at higher level for longer period in treatments. It appears that

· tremendously by delaying the senescence.

Initial increase in the fresh weight of spikes in both the vase solution (treated and control) can be attributed to the increased requirement of the spikes for opening of flowers. The decline in fresh weight is due to less water uptake, high respiration rate and also membrane leakage. In the absence of current photoassimilates, reserved carbohydrates are used in respiration. When fresh weight of flower spikes declined below its initial fresh weight then the flower spikes are said to have lost their vase life quality and this is treated as vase life. The vase life was extended significantly with the treatment of lipoic acid.

Membrane stability index (MSI %) represents the ,change in the permeability 'of membrane. Reduction in MSI indicates status of ion leakage or solute leakage. There is a sharp decline in MSI in both the varieties . Membrane deterioration leads to the loss of intracellular compartmentalization, an important feature of senescence. This results in ion and water leakage (cells are not able to retain required ions and water). Membrane stability and fluidity is also lost by the direct attack of free radicals on lipids and protein the constituents of bilayer. MSI was however alleviated by LA treatment.

The thiobarbituric acid reactive substances (TBARS) increased with the senescence indicating increased level of lipid peroxidation. TBARS content was less in LA treated flowers in both the varieties in comparison to control. The LA could delay the senescence by delaying lipid peroxidation (through reduction in TBARS) and maintain higher MSI for the longer duration -Thus LA is beneficial against oxidative lipid damage .

Lipoxygenase are a class of enzymes, which catalyze the hydroperoxidation of polyunsaturated fatty acids, especially in cell membrane. Lipoxygenase activity increases during the flower senescence. Thus, an increase in LOX activity represents an increase in the cell membrane permeability or reduction in MSI. There is a strong temporal correlation during senescence between

50

changes in LOX activity and superoxide radical production by the membranes. From the less LOX activity in LA treated flowers it can be conceived that the free radicals produced through membrane associated LOX may be scavenged by LA. This may contribute to some extent for the extension of vase life of spikes held in LA solution,

Plants have evolved wide range of mechanism to counteract the oxidative stress. Cellular damage caused by lipid peroxidation might be reduced or prevented by protective mechanism involving free radical and peroxide scavenging enzymes such as Superoxide dismutase(SOD), catalase (CAT), glutathione reductase, ascorbate peroxidase etc.

SOD is generally considered key enzyme in the regulation of intracellular concentration of superoxide radical (02.-) and peroxide which can react in Haber-Weiss reaction to form hydroxyl radical leading to peroxidation. Initially activity of SOD is slightly increased then there was a decline at later stage during senescence. SOD activity was higher and retained for longer duration in the flowers treated by LA.

Hydrogen peroxide-CH20) produced during the dismutation of superoxide radicals by SOD is scavenged by enzyme catalase. Activity of catalase is initially increased but declined at latter stages of senescence. Catalase activity was retained at higher level in the flowers treated with LA in comparison to control in both the cultivars. This is because LA directly scavenges free radicals.

Glutathione reductase (GR) catalyses the reduction of glutathione disulphide with the accompanying oxidation of NADPH. GR activity is decreased as the senescence proceeds after initial increase. Reduction in GR activity results in ~

decline in the level of reduced glutathione, known to be an important factor in preventing oxidative injuries. The LA supplemented gladiolus flowe rs showed increased GR activity over control. Hence it can be conceived that LA delayed the senescence by increasing/retaining GR activity for longer period.

Membrane bound ascorbate peroxidase (AP) is found to scavenge the H202which is produced by

the activity of SOD on the superoxide radical. The ascorbate activity was found to be directly correlated with the reduction in free radical induced damage. LA treated flowers showed enhanced level of AP even at later stages of senescence over the control. Thus increased level of AP may also be one of the reason s in postponing the senescence by scavenging H202and preventing membrane damage.

On the basis of this study it is clear that LA can increase or retain the activity of SOD, CAT, GR, and AP thereby increasing antioxidant status of these enzymes to is scavenge free radicals produced during senescence. Apart from this LA can reduce lipid peroxidation by decreasing lipoxygenase activity and TBARS contents which in turn prevent or delay the membrane damage to some extent which ultimately leads to the delay in flower senescence. Thus LA acts as a free radical scavenger and hence delayed the flower senescence in gladiolus flowers .

There was a decline in protein content during senescence. Flower senescence is mainly associated with the loss of protein. The protein content is reduced due to little de novo synthesis and considerable protein degradation It is understood that free radicals attack amino acid residues of proteins causing conformational changes in proteins causing them to be recognized by specific proteases for degradation.Also a change in membrane lipid composition induces conformational change s in proteins , by providing an unfavorable environment deleteriously affecting the activity of key enzymes. (The proteins are excluded into liquid crystalline domain from gel phase liquid domain which progressively increases during senescence. Proteins trapped in the gel phase are degraded by proteases. It is observed that LA treated flowers maintained higher protein over control. It may be because LA acts as a free radical scavenger.

(Arora et al., 2002; Singh and Jegadheesan, 2003; Srivastava, 2003; Arora, 2007a; Arora, 2007b. In press).

51

D. 5-Su lp hosa li cyli c acid delayed the senescence and increased the vase life of gladiolus flowers: Hundred ppm of 5­sulphosalicylic acid (5-SSA) along with 2 % sucrose in holding solution delayed the senescence of gladiolus flowers and consequen tly increased vase life up to 11.2 days while in control it was 5.3 days. The flower quality was improved in terms of percentage flowering , fresh weight and water uptake. Treated flowers also exhibited delayed and reduced ion leakage as well as respiration rate. Superoxide dismutase (SOD) and catalase (CAT) activities were maintained at higher level for longer time in treated flowers to scavenge free radicals. Thiobarbituric acid reactive substances (TBARS) content and lipoxygenase (LOX) activity were also kept in check by 5-SSA thus reducing the level of peroxidation. It is proposed and proved that 5-SSA delayed senescence by acting as a free radical scavenger (Ezhilmathi et al., 2007).

E. Role of polyamines in flower senescence: Polyamines (PA) are now regarded as a new class of growth substances and are well known for their antisenescence effects on animal and plant tissue. The polyamines-spermidine and spermine occur ubiquitously in the plant kingdom, together with their diamine precursor putrescine.

Treatment with either of 100 ppm i.e. spermine, spermidine and putrescine along with 2 % sucrose solution increased the vase life to 9.6 and 8.4 days in Dhanwantari and Snow Princess, respectively by spermine and then followed by spermidine, putrescine and control. Spermine and spermidine could successfully extend vase life of flowers by maintaining higher MSI, protein, higher uptake of vase solution and thus maintaining higher fresh weight for longer duration. Spermine and spermidine were able to reduce the activity of lipoxygenase enzyme and lipid peroxidation that are responsible for membrane deterioration and loss of com partmentalization. Also, during the senescence activi ties of various antioxi dant enzymes viz. SOD , CAT, glutat hione redu ctase (GR) and ascorbate peroxidase (AP) were at higher level for longer period in treatments. It appears that

spermine and spermidine acted as free radical scavengers and consequently delayed the flower senescence. Spermine was found to be more effective than spermidine in prolonging the process-. . of flower senescence.

be negatively correlated with flower senescence when measured as reduction in fresh weight. Sodium benzoate (0.1 mM ) a known free radical scavenger could delay the sene scence of flowers significantly by sustaining the MSI for longer time

J. Abscisic acid is the hormonal trigger for cascade of degradative changes in ethylene insensitive gladiolus flowers: The senescence of flower petals is as sociated with a series of ~hysiological and biochemical changes. This

with ABA. !he ability of ABA was inhibited by t~eatment with GA a hormone which extends the

3

hfe of flowers. This suggests that effect of GA could in part be due to its antagonistic ability of endogenous ABA to cause sene scence (Divisional

Quantitative isoform analysis revealed presence oftwo isoforms viz., Cu/Zn SOD and Fe-SOD both of which showed a similar activity in response to spermine treatment. The pattern of activity was same as that of total SOD activity. SOD isozyme analysis by negative staining re vealed three isoforms two of which were found to be Cu/Zn isoform while one was a Fe-SOD. Spermine treatment enhanced activity of all three isoforms particularly that of smaller Cu/Zn SOD. Analysis of RT-PCR products on agarose gel revealed a single band . A SOD cDNA of approximately 330 bp"was amplified by degenerate primers.

(Singh et al. , 2005b; Singh et al., 2005c; Singh and Srivastava, 2006).

F. 8-Hydroxy quinoline (8-HQ) improved post­harvest quality of gladiolus cut spikes: Treatment with antimicrobial agent 8-HQ (300 ppm) with 5 % sucrose improved the keeping quality of cut spikes. This treatment significantly enhanced the percent gain in fresh and dry weight of cut spikes as well as recorded high reducing and non reducing sugars, carotenes and anthocynin pigments in the petals on, 4 th day after treatment (OAT). 8-HQ treatment maintained higher activities of SOD and GR reduced lipoxygenase activity and lipid peroxidation in petal s at 5 OAT. All these factors contributed towards better membrane integrity exhibited as high MSI, which delayed the petal senescence and doubl ed the vase life and improved the flower quality of gladiolus cut spikes (Singh et al. , 2005a). .

G. Amelioration of membrane damage during flower senescence by sodium benzoate: Role of membranes integrity during flower sene scence of gladiolus and its alleviation by sodium benzoate was studied. Membrane integrity in terms MSI was positively related with delay in senescence while, lipid peroxidation and LOX activity were found to

at higher level in comparison to control and restricting the TBARS content and LOX activity (Singh, 2005) .

H. Some other chemicals which improve vase life: Experiments revealed that boric acid (250 ppm) , glycerol (l 00 ppm) , salicylic acid (100 ppm ) and dimethyl sulphoxide (600 ppm) along with 2 % sucro se in holding solution were able to increase the vase life of cut gladiolus flowers by 16, 31, 45 and 45 % over the control. The number of opened florets was highest in salicylic acid treatment followed by glycerol boric acid and dimethyl sulphoxide.

I. Polyols regulate the flower senescence by delaying programmed cell death in gladiolus: The best treatment for extending the vase life and quality was treatment of flower spike with 75 mM of inositol followed by trehalose (l00 mM), mannitol (100 mM), and sorbitol (l00 mM). The vase life of cut flower spikes was significantly increased by the polyol treatments ranging from 14 % with sorbitol alliI 38 % with inositol against control. The flower quality was improved in terms of percentage flowering, fresh weight and water uptake. Treated flowers also exhibited delayed and reduced lipoxygenase enzyme activity as well as ion leakage. In the present study, membrane di sruption and DNA fragmentation, e ve nts characteristic of PCD , were found to be present in the advanced stage of petal senescence indic ating that plant and animal cell death phenomena share one of the molecular events in the execution phase. When the gladiolus flowerets were treated with inositol both wilting and DNA fragmentation of petal s were suppressed/delayed . This pr~vides the initial evidence that inositol has an inhibitory / suppressi ve effect on apoptoti c cell death (Arora and Singh, 2006; Arora, 2007 b. In press) .

includes .an increase in hydrolytic en zymes, degradation of macromolecules , increased re spiratory activity and lo ss of cellular compartmentalization due to leakiness of cell membrane. Invol vement of ethyl ene in triggering senescence in gladiolus is unlikely as this is an ethylene insensitive flower. The signals that initiate degradative processes during senescence are largely unknown for gladiolus. Abscisic acid (ABA) may be a possible candidate for hormonal trigger for death of flowers such as gladiolus that do not respond.to ethylene. Experiments were conducting to elucidate th e role of ab sci si c acid in the senescence of ethylene in sensitive gladiolus flower s. ABA advanced the senescence of gladiolus flowers through reduction in daily vase solution uptake, fresh weight of spikes and flower diameter in a concentration dependent manner in both the cultivars. Exogenous ABA prematurely up regulates events that occur during natural senescence, (control) such as loss of differential membrane permeability, increase in lipid peroxidation and induction of protease activity. Endogenous ABA content increased with th e advancement of senescence during natural senescence in control. !he c.oncentration of ABA was increased by imposing osmotic stres s using 0.3 M sorbitol to prove the role of endogenous ABA on flower senescence. Flowers were kept in 0.3 M sorbitol, a concentration that decreases but does not eliminate water uptake. Endogenous ABA increases before flower opening and continue to increase during petal senescence. An osmotic stress by sorbitol not only increases endogenous level s of but also up regulates the same parameters of senescence as those occurring during natural senescence and after application of exogenous ABA. To generate further evidence for the importance of ABA we inhibited its action in petals by adding GA (a well known

. 3 antagoni st ofABA function) to vase solution along

Annual Report, 2006-2007. In press)

K. Molecular characterization and regulation of senescence in gladiolus

Since the initial di scovery of ETRI in Arabidopsis, many homologues have been isolated from plants but all belong to a class of climacteric or ethylene sensitive plants. To identify which processe s in Gladiolus, ethylene-insensitive flowers, are associated with changes in ethylene perception, we clone and characterized the gladiolus homologue of the gene encoding the ethylene receptors. PCR amplifications using degenerate oligonucleotide primers resulted in the isolation of partial cDNAs of the expected size for fragment of GgERSl (1.0 kbp) . The amplified fragment were cloned' into the pGEM-T vector, sequenced and found to have strong nucleotide sequence similarity to the Arabidopsis AtERSl mRNA. We isolated two gladiolus (Gladiolus grandiflora cv . Traveler) full-length cDNA homologues of the Arabidopsis ethylene receptor genes ERSI by 5' and 3' random amplification of cDNA end s (RACE-PCR) and designated them as GgERSla and GgERSlb. (We reported these kind s of receptors first time from any kind of plant)

Genomic organization of ethylene receptors was performed by southern analysis and the results suggested that gladiolus is having at least two ethylene receptors homologs and are members of a gene family. By western blot analy sis, we could able to detect both the ethylene receptors viz. , GgERSla and GgERSlb proteins as expected size of70.6 KD and 34.9 KD, respectively, as anti-Cm­ERSI-KD antibodies could cross-react with gladiolus GgERSl receptors. The expression studies (mRNA and protein) clearly shown that both the gene s are expressing well, the expression of both the genes increase the expectation that both the gene is important for the subfunctionalization

, ~

52 53

spermine and spermidine acted as free radical scavengers and consequently delayed the flower senescence. Spermine was found to be more effective than spermidine in prolonging the process-. . of flower senescence.

be negatively correlated with flower senescence when measured as reduction in fresh weight. Sodium benzoate (0.1 mM ) a known free radical scavenger could delay the sene scence of flowers significantly by sustaining the MSI for longer time

J. Abscisic acid is the hormonal trigger for cascade of degradative changes in ethylene insensitive gladiolus flowers: The senescence of flower petals is as sociated with a series of ~hysiological and biochemical changes. This

with ABA. !he ability of ABA was inhibited by t~eatment with GA a hormone which extends the

3

hfe of flowers. This suggests that effect of GA could in part be due to its antagonistic ability of endogenous ABA to cause sene scence (Divisional

Quantitative isoform analysis revealed presence oftwo isoforms viz., Cu/Zn SOD and Fe-SOD both of which showed a similar activity in response to spermine treatment. The pattern of activity was same as that of total SOD activity. SOD isozyme analysis by negative staining re vealed three isoforms two of which were found to be Cu/Zn isoform while one was a Fe-SOD. Spermine treatment enhanced activity of all three isoforms particularly that of smaller Cu/Zn SOD. Analysis of RT-PCR products on agarose gel revealed a single band . A SOD cDNA of approximately 330 bp"was amplified by degenerate primers.

(Singh et al. , 2005b; Singh et al., 2005c; Singh and Srivastava, 2006).

F. 8-Hydroxy quinoline (8-HQ) improved post­harvest quality of gladiolus cut spikes: Treatment with antimicrobial agent 8-HQ (300 ppm) with 5 % sucrose improved the keeping quality of cut spikes. This treatment significantly enhanced the percent gain in fresh and dry weight of cut spikes as well as recorded high reducing and non reducing sugars, carotenes and anthocynin pigments in the petals on, 4 th day after treatment (OAT). 8-HQ treatment maintained higher activities of SOD and GR reduced lipoxygenase activity and lipid peroxidation in petal s at 5 OAT. All these factors contributed towards better membrane integrity exhibited as high MSI, which delayed the petal senescence and doubl ed the vase life and improved the flower quality of gladiolus cut spikes (Singh et al. , 2005a). .

G. Amelioration of membrane damage during flower senescence by sodium benzoate: Role of membranes integrity during flower sene scence of gladiolus and its alleviation by sodium benzoate was studied. Membrane integrity in terms MSI was positively related with delay in senescence while, lipid peroxidation and LOX activity were found to

at higher level in comparison to control and restricting the TBARS content and LOX activity (Singh, 2005) .

H. Some other chemicals which improve vase life: Experiments revealed that boric acid (250 ppm) , glycerol (l 00 ppm) , salicylic acid (100 ppm ) and dimethyl sulphoxide (600 ppm) along with 2 % sucro se in holding solution were able to increase the vase life of cut gladiolus flowers by 16, 31, 45 and 45 % over the control. The number of opened florets was highest in salicylic acid treatment followed by glycerol boric acid and dimethyl sulphoxide.

I. Polyols regulate the flower senescence by delaying programmed cell death in gladiolus: The best treatment for extending the vase life and quality was treatment of flower spike with 75 mM of inositol followed by trehalose (l00 mM), mannitol (100 mM), and sorbitol (l00 mM). The vase life of cut flower spikes was significantly increased by the polyol treatments ranging from 14 % with sorbitol alliI 38 % with inositol against control. The flower quality was improved in terms of percentage flowering, fresh weight and water uptake. Treated flowers also exhibited delayed and reduced lipoxygenase enzyme activity as well as ion leakage. In the present study, membrane di sruption and DNA fragmentation, e ve nts characteristic of PCD , were found to be present in the advanced stage of petal senescence indic ating that plant and animal cell death phenomena share one of the molecular events in the execution phase. When the gladiolus flowerets were treated with inositol both wilting and DNA fragmentation of petal s were suppressed/delayed . This pr~vides the initial evidence that inositol has an inhibitory / suppressi ve effect on apoptoti c cell death (Arora and Singh, 2006; Arora, 2007 b. In press) .

includes .an increase in hydrolytic en zymes, degradation of macromolecules , increased re spiratory activity and lo ss of cellular compartmentalization due to leakiness of cell membrane. Invol vement of ethyl ene in triggering senescence in gladiolus is unlikely as this is an ethylene insensitive flower. The signals that initiate degradative processes during senescence are largely unknown for gladiolus. Abscisic acid (ABA) may be a possible candidate for hormonal trigger for death of flowers such as gladiolus that do not respond.to ethylene. Experiments were conducting to elucidate th e role of ab sci si c acid in the senescence of ethylene in sensitive gladiolus flower s. ABA advanced the senescence of gladiolus flowers through reduction in daily vase solution uptake, fresh weight of spikes and flower diameter in a concentration dependent manner in both the cultivars. Exogenous ABA prematurely up regulates events that occur during natural senescence, (control) such as loss of differential membrane permeability, increase in lipid peroxidation and induction of protease activity. Endogenous ABA content increased with th e advancement of senescence during natural senescence in control. !he c.oncentration of ABA was increased by imposing osmotic stres s using 0.3 M sorbitol to prove the role of endogenous ABA on flower senescence. Flowers were kept in 0.3 M sorbitol, a concentration that decreases but does not eliminate water uptake. Endogenous ABA increases before flower opening and continue to increase during petal senescence. An osmotic stress by sorbitol not only increases endogenous level s of but also up regulates the same parameters of senescence as those occurring during natural senescence and after application of exogenous ABA. To generate further evidence for the importance of ABA we inhibited its action in petals by adding GA (a well known

. 3 antagoni st ofABA function) to vase solution along

Annual Report, 2006-2007. In press)

K. Molecular characterization and regulation of senescence in gladiolus

Since the initial di scovery of ETRI in Arabidopsis, many homologues have been isolated from plants but all belong to a class of climacteric or ethylene sensitive plants. To identify which processe s in Gladiolus, ethylene-insensitive flowers, are associated with changes in ethylene perception, we clone and characterized the gladiolus homologue of the gene encoding the ethylene receptors. PCR amplifications using degenerate oligonucleotide primers resulted in the isolation of partial cDNAs of the expected size for fragment of GgERSl (1.0 kbp) . The amplified fragment were cloned' into the pGEM-T vector, sequenced and found to have strong nucleotide sequence similarity to the Arabidopsis AtERSl mRNA. We isolated two gladiolus (Gladiolus grandiflora cv . Traveler) full-length cDNA homologues of the Arabidopsis ethylene receptor genes ERSI by 5' and 3' random amplification of cDNA end s (RACE-PCR) and designated them as GgERSla and GgERSlb. (We reported these kind s of receptors first time from any kind of plant)

Genomic organization of ethylene receptors was performed by southern analysis and the results suggested that gladiolus is having at least two ethylene receptors homologs and are members of a gene family. By western blot analy sis, we could able to detect both the ethylene receptors viz. , GgERSla and GgERSlb proteins as expected size of70.6 KD and 34.9 KD, respectively, as anti-Cm­ERSI-KD antibodies could cross-react with gladiolus GgERSl receptors. The expression studies (mRNA and protein) clearly shown that both the gene s are expressing well, the expression of both the genes increase the expectation that both the gene is important for the subfunctionalization

, ~

52 53

process for providing ethylene insensitivity to gladiolus flowers. Presently working on functional analysis of gladiolus ethylene receptors.

(Arora, 2005; Arora et al., 2006) .

L. Isolation and molecular characterization of cysteine protease gene in gladiolus: Senescence is the final event in the life of many plant tissues and is a highly regulated process that involves structural, biochemical and molecular changes that in many cases bear the hallmarks of programmed cell death. Most of the information on the biology of flower senescence has been obtained from the study of flowers like carnation and morning glory where the gaseous plant hormone ethylene coordinates the entire process. Ethylene is not always the primary coordinating agent in ripening arid senescence. Although much is known about ethylene-mediated senescence in fruits and flowers, there has been very little report describing the molecular basis of non-climacteric ripening of fruits or the ethylene-insensitive senescence of flowers. Gladiolus flowers are not usually regarded as being ethylene-sensitive. Petals from this species were therefore used in the present study as a system in which to investigate events associated with protein degradation during ethylene-independent floral senescence. A putative cysteine protease (GgCyP) gene was cloned from Gladiolus using degenerate PCR primers and the expression pattern of this gene was determined semi-quantitatively by RT-PCR. There was a dramatic increase in the expression of GgCyP indicating that this gene may encode an important enzyme for the proteolytic process in this species (Arora and Ezura, 2003; Arora and Singh , 2004).

M. Differential regulation of cysteine protease and ethylene receptor genes by salicylic acid in Gladiolus flowers: We cloned and characterized the gladiolus homologue of the gene encoding the ethylene receptors (GgERSla & GgERSlb). A putative cysteine protease (GgCyP) gene was also cloned from Gladiolus using degenerate PCR primers and RACE- PCR techniques. Salicylic acid (SA) is mainly known for its role in plant defense mechan ism-it is a potent inducer of cell death.

However, recent studies on defense related mutants of Arabidopsis provide evidence that SA regulates cell expansion and cell division, as well as cell death, suggesting a role in the balance between growth and senescence. SA may have a general homeostatic role in plant development. On the basis of results from expression profiles we proposed a model for differential regulation of cysteine protease and ethylene receptor genes by SA and delay the senescence of ethylene-insensitive gladiolus flowers (Arora and Ezura, 2003; Arora and Singh, 2004; Arora, 2005; Arora et al., 2006).

N. Detection of ethylene receptor protein GgERSla and GgERSlb: To elucidate the expression of both the GgERS1a and GgERSlb proteins in gladiolus flower, the antibodies against Cm-ERS1 protein were used, as the sequence homology between melon polypeptide regions K1l7-D327 (Cm-ERS1KD), chosen as an antigen, showed very high homology (80 %) with gladiolus region. This region lies between the transmembrane and histidine kinase domains for the polypeptide chain of Cm-ERSl as well as in GgERS1 protein. For detection of native GgERS1 protein, first stage flower development was harvested, and proteins were extracted. The solubilized fraction was used as the microsomal membrane fraction. The protein concentrations in the total, soluble, and microsomal membrane protein samples were determined. By western blot analysis, we could able to detect both the ethylene receptors viz., GgERS1a and GgERS1b proteins as expected size of70.6 KD and 34.9 KD, respectively, as anti-Cm-ERS1-KD antibodies could cross-react with gladiolus GgERSl receptors. The expression of both the genes at protein levels also increases our expectation that both the gene is important for the subfunctionalization process (Arora, 2005).

O. Gladiolus ethy lene r eceptors sub-cellular localization: The ethylene receptor ERS I has a modular structure and contains three predicted transmembrane segments implicated in both membrane localization and ethylene binding. We analyze the localization of GgERSl in tobacco as well as in onion membranes by reporter protein

54

GFP. A fusion construct between GgERSl and green fluorescent protein (GFP) was made by DNA coding for GgERSl was amplified from its cDNA by PCR, which removed the stop codon and introduced Sall restriction sites. The Sall restriction site was also used just before the initiation codon of the GgERS1. The amplified GgERSJ coding sequence was digested and cloned into sGFP (S65T) plasmid, a pUC-based vector containing CaMV35S-sGFP (S65T)-NOS3' that was kindly provided by Dr. Yasuo Niwa (University of Shizuoka, Japan). Tobacco leaves were cut from plants grown in a growth room, and placed in Petri dishes containing a solid MS medium. Leaf samples were then bombarded with gold particles (1 urn) that were coated with 10 ug of plasmid DNA at a distance of 9 ern, using a BioRad PDS-lOOO/He Particle Delivery System at 900 psi He pressure under a vacuum of 28 in. Hg. After bombardment, the tobacco leaves petioles were placed in the medium, and samples were incubated at 25 "C in the dark. After 20-24 h, the leaves were examined for GFP fluorescence. Leaf pieces were sliced with a razor blade and placed on glass slides; water was added to the adaxial surfaces, and glass cover slips were placed over the slices. Cells were examined using a confocal microscope (Leica TCS SP2, Leica Microsystem, Wetzlar, Germany). Excitation wavelengths were set at 488 nm and 568 nm, and images were collected through an FITC filter for GFP fluorescence (green channel) and through a TRITC filter for chlorophyll fluorescence (red channel). All images were collected through a water immersi on objective (x 63, 1.2 numerical aperture). Optical sections were 0.30 urn thick. Final image assembly was done with Adobe Photoshop 6.0 software. Only the green channel images are shown in the results in order to allow the GFP subcellular localization patterns to be seen more clearly. Leaf vein epidermal cells or trichomes were observed for subcellular localization. All images were taken from the surface closest to the cell wall. In control cells expressing GFP alone, fluorescence was observed in both the cytoplasm and nucleus, which is typical for GFP distribution. On the other hand,

55

cells expressing GgERS I-GFP showed fluorescence along a reticular network that displayed characteristic three-way junctions with angles of approximately 120 0 between the branches, which is identical to the pattern generated by an ER-targeted mGFP5-ER fusion protein containing an N-terminal signal peptide and a C­terminal HDEL motif. These results suggest that the GgERSl contain determinants for ER localization.

III. Future prospects for post-harvest physiology of fruits

During the last 20 years, advances have been made not only in understanding the biological and environmental factors influencing senescence, ripening and deterioration of harvested commodities. However, much more research and development is needed in further improving the she1flife and quality aspects ofdifferent perishable commodities in future. Some of the most important future research priorities are being presented here under different heads:

A. Growth and development

• To determine the exact role of plant hormones especially other than ethylene (abscisic acid, auxins, gibberellins, cytokinins and the polyamines) in fruit development and ripening and how best it could be exploited at basic and applied levels.

• Role of developing seeds inside the fruit and their interaction with the overall growth, development and ripening of the fruit is an interesting area of research.

• Productivity and quality of temperate fruits will be drastically affected by global climate change. So, there is need for research to diminish the adverse effects and enhance the long-term adaptability of fruit plants towards the changing climate.

• Anatomical features need to be investigated in details with their possible correlations to the ripening/ripening linked processes and changes.

process for providing ethylene insensitivity to gladiolus flowers. Presently working on functional analysis of gladiolus ethylene receptors.

(Arora, 2005; Arora et al., 2006) .

L. Isolation and molecular characterization of cysteine protease gene in gladiolus: Senescence is the final event in the life of many plant tissues and is a highly regulated process that involves structural, biochemical and molecular changes that in many cases bear the hallmarks of programmed cell death. Most of the information on the biology of flower senescence has been obtained from the study of flowers like carnation and morning glory where the gaseous plant hormone ethylene coordinates the entire process. Ethylene is not always the primary coordinating agent in ripening arid senescence. Although much is known about ethylene-mediated senescence in fruits and flowers, there has been very little report describing the molecular basis of non-climacteric ripening of fruits or the ethylene-insensitive senescence of flowers. Gladiolus flowers are not usually regarded as being ethylene-sensitive. Petals from this species were therefore used in the present study as a system in which to investigate events associated with protein degradation during ethylene-independent floral senescence. A putative cysteine protease (GgCyP) gene was cloned from Gladiolus using degenerate PCR primers and the expression pattern of this gene was determined semi-quantitatively by RT-PCR. There was a dramatic increase in the expression of GgCyP indicating that this gene may encode an important enzyme for the proteolytic process in this species (Arora and Ezura, 2003; Arora and Singh , 2004).

M. Differential regulation of cysteine protease and ethylene receptor genes by salicylic acid in Gladiolus flowers: We cloned and characterized the gladiolus homologue of the gene encoding the ethylene receptors (GgERSla & GgERSlb). A putative cysteine protease (GgCyP) gene was also cloned from Gladiolus using degenerate PCR primers and RACE- PCR techniques. Salicylic acid (SA) is mainly known for its role in plant defense mechan ism-it is a potent inducer of cell death.

However, recent studies on defense related mutants of Arabidopsis provide evidence that SA regulates cell expansion and cell division, as well as cell death, suggesting a role in the balance between growth and senescence. SA may have a general homeostatic role in plant development. On the basis of results from expression profiles we proposed a model for differential regulation of cysteine protease and ethylene receptor genes by SA and delay the senescence of ethylene-insensitive gladiolus flowers (Arora and Ezura, 2003; Arora and Singh, 2004; Arora, 2005; Arora et al., 2006).

N. Detection of ethylene receptor protein GgERSla and GgERSlb: To elucidate the expression of both the GgERS1a and GgERSlb proteins in gladiolus flower, the antibodies against Cm-ERS1 protein were used, as the sequence homology between melon polypeptide regions K1l7-D327 (Cm-ERS1KD), chosen as an antigen, showed very high homology (80 %) with gladiolus region. This region lies between the transmembrane and histidine kinase domains for the polypeptide chain of Cm-ERSl as well as in GgERS1 protein. For detection of native GgERS1 protein, first stage flower development was harvested, and proteins were extracted. The solubilized fraction was used as the microsomal membrane fraction. The protein concentrations in the total, soluble, and microsomal membrane protein samples were determined. By western blot analysis, we could able to detect both the ethylene receptors viz., GgERS1a and GgERS1b proteins as expected size of70.6 KD and 34.9 KD, respectively, as anti-Cm-ERS1-KD antibodies could cross-react with gladiolus GgERSl receptors. The expression of both the genes at protein levels also increases our expectation that both the gene is important for the subfunctionalization process (Arora, 2005).

O. Gladiolus ethy lene r eceptors sub-cellular localization: The ethylene receptor ERS I has a modular structure and contains three predicted transmembrane segments implicated in both membrane localization and ethylene binding. We analyze the localization of GgERSl in tobacco as well as in onion membranes by reporter protein

54

GFP. A fusion construct between GgERSl and green fluorescent protein (GFP) was made by DNA coding for GgERSl was amplified from its cDNA by PCR, which removed the stop codon and introduced Sall restriction sites. The Sall restriction site was also used just before the initiation codon of the GgERS1. The amplified GgERSJ coding sequence was digested and cloned into sGFP (S65T) plasmid, a pUC-based vector containing CaMV35S-sGFP (S65T)-NOS3' that was kindly provided by Dr. Yasuo Niwa (University of Shizuoka, Japan). Tobacco leaves were cut from plants grown in a growth room, and placed in Petri dishes containing a solid MS medium. Leaf samples were then bombarded with gold particles (1 urn) that were coated with 10 ug of plasmid DNA at a distance of 9 ern, using a BioRad PDS-lOOO/He Particle Delivery System at 900 psi He pressure under a vacuum of 28 in. Hg. After bombardment, the tobacco leaves petioles were placed in the medium, and samples were incubated at 25 "C in the dark. After 20-24 h, the leaves were examined for GFP fluorescence. Leaf pieces were sliced with a razor blade and placed on glass slides; water was added to the adaxial surfaces, and glass cover slips were placed over the slices. Cells were examined using a confocal microscope (Leica TCS SP2, Leica Microsystem, Wetzlar, Germany). Excitation wavelengths were set at 488 nm and 568 nm, and images were collected through an FITC filter for GFP fluorescence (green channel) and through a TRITC filter for chlorophyll fluorescence (red channel). All images were collected through a water immersi on objective (x 63, 1.2 numerical aperture). Optical sections were 0.30 urn thick. Final image assembly was done with Adobe Photoshop 6.0 software. Only the green channel images are shown in the results in order to allow the GFP subcellular localization patterns to be seen more clearly. Leaf vein epidermal cells or trichomes were observed for subcellular localization. All images were taken from the surface closest to the cell wall. In control cells expressing GFP alone, fluorescence was observed in both the cytoplasm and nucleus, which is typical for GFP distribution. On the other hand,

55

cells expressing GgERS I-GFP showed fluorescence along a reticular network that displayed characteristic three-way junctions with angles of approximately 120 0 between the branches, which is identical to the pattern generated by an ER-targeted mGFP5-ER fusion protein containing an N-terminal signal peptide and a C­terminal HDEL motif. These results suggest that the GgERSl contain determinants for ER localization.

III. Future prospects for post-harvest physiology of fruits

During the last 20 years, advances have been made not only in understanding the biological and environmental factors influencing senescence, ripening and deterioration of harvested commodities. However, much more research and development is needed in further improving the she1flife and quality aspects ofdifferent perishable commodities in future. Some of the most important future research priorities are being presented here under different heads:

A. Growth and development

• To determine the exact role of plant hormones especially other than ethylene (abscisic acid, auxins, gibberellins, cytokinins and the polyamines) in fruit development and ripening and how best it could be exploited at basic and applied levels.

• Role of developing seeds inside the fruit and their interaction with the overall growth, development and ripening of the fruit is an interesting area of research.

• Productivity and quality of temperate fruits will be drastically affected by global climate change. So, there is need for research to diminish the adverse effects and enhance the long-term adaptability of fruit plants towards the changing climate.

• Anatomical features need to be investigated in details with their possible correlations to the ripening/ripening linked processes and changes.

B. Ethylene biosynthesis, receptors and signal transduction

• More research is required to discover the developmental factors and regulatory genes that control the expression of ethylene biosynthesis genes.

• The only gene and enzyme that has not been fully understood in the ethylene biosynthesis pathway is related to the conversion of ACC into MACe. So, complete characterization of ACC­N malonyl transferase at gene and enzyme levels is important in view of its regulatory role in ethylene biosynthesis by diverting ACC from its normal route to ethylene.

• Identification of additional components involved in ethylene signal transduction, their J ur ther characterization and studies on the biochemistry of ripening are still essential for complete understanding of the climacteric ripening processes. This will also help us in understanding the process in vol ved in differential perception of ethylene during development as even today it is not well understood.

C. Climacteric and/versus non-climacteric ripening

• Recent work on the molecular basis of developmental ripening control suggested common regulators for climacteric and non­climacteric ripening. The work further indicated that the ripening of climacteric fruits comprises a portion of non-climacteric behaviour. So, an exhaustive identification of ethylene-dependent and ethylene-independent gene expression remains to be done. Further, little is known about the regulation of ripening in non-climacteric fruit and the upstream regulation of ethylene in climacteric fruits ..

• What is the exact reason and specific role of rise in respiration (climacteric rise) during ripening of climacteric fruits? During ripening of mango and tomato fruits alternate respiration (cyanide resistant respiration) is the major

portion of total respiration . Its role, significance and manipulation in slowing down the ripening process need the attention.

D. Storage

• Identification of threshold values of water loss that induce hormonal changes; various other physiological, biochemical and biophysical responses; noticeable shriveling an d physiological disorders .

• How to minimize the deterioration at quality levels during the prolonged storage of fruits (not sensitive to chilling injuries) at low temperature such as apple?

E. Physiological disorders

• Post-harvest disorders having phy siological reasons need to be studied in view of regulating the requirements which can decrease the development of such physiological disorders. Usually; temperature, a/co

2 ratio, rate of

ventilation, mineral content in tissue, pre-harvest factors, carbohydrate metabolism, and oxidative changes are involved for such disorders. Thus, understanding the physiological basis of such disorders will help in overcoming the losses by taking required pre-harvest and post-harvest precautions.

• Understanding the reasons for inter-cultivar differences in susceptibility to vari ous physiological disorders including chilling injury. Investigations on the physiological and biochemical basis of controlled atmosphere­induced physiological disorders and how these could be minimized during storage. Further, optimizing nutrition and endogenous hormonal balance to reduce the incidence and severity of various physiological disorders ne ed the attention.

F. Traditional approach, mutants and transgenic

• Development of rapid, reliable, repeatable and efficient regeneration and transformation protocols of fruit plants including the woody species.

• In future, technology or the availability of methodology would not be a limiting factor but, scope of genetically engineered fruits with suitable traits would more depend on economics and consumers' preference.

• It would be better that instead of interfering with whole of the ripening process, attention should be to target very specific proce sses such as, the fruit colour development or the kinetics of sugar accumulation in fruits.

• Private companies are too eager to cash in on the new technologies/transgenics and so many new products are being developed without a full understanding of the metabolic processes taking place especially at the levels of regulation and the direct, indirect and subsequent effects of introduced change. Thereby, we are running the risk of developing products with unintended but potentially adverse agronomic, adaptable and acclimatizing characteristics at whole plant or/ and crop levels.

• For economically backward and developing countries molecular and biotechnological research should not be at the expense of traditional and other innovative approaches of crop improvement and other already well established disciplines of agricultural sciences.

G. New and innovative ways and means to control ripening

• To discover, invent or synthesis the novel compounds preferably volatile but non-toxic in nature that could either inhibit or delay the ripening process when applied to pre or post harvested fruits.

• Ethanol is a promising, cost-effective and non­toxic chemical that can delays the ripening in tomato fruits . This therefore opened up the research to further standardize the dose of ethanol which can effectively delay the ripening irrespective of variety, for different durations and for fruits at different ripening stages so that the required basic information can be generated for the large scale application of ethanol to delay the ripening.

• Reproductive physiology in terms oftiming of floral induction, rate of flower development, extent of flowering, fruit growth, and developmental, incompatibility, sterility, parthenogenesis, parthenocarpy and apomixes (including the method of micropropagation) is not fully exploited at commercial scale in India for improving overall production of fruits , vegetable and flowers.

• Precise understanding of physiological basis for flower and pre-mature fruit drop will also contribute significantly towards enhancing the productivity and production.

IV. Future prospects for post-harvest physiology of flower senescence

There is little doubt that the molecular and genetic analyses of flower senescence made in the past 5 years have raised our awareness of the complex interactions that occur to regulate flower development and senescence. Genetic technologies have enabled scientists to search for senescence­related genes in plants often described as science models (e.g., Petunia, Arabidopsisi, and then translate the data into other species to determine the functional significance of the expression of specific genes in specific tissues after harvest. Interactions between ethylene, cytokinin, sugars and various hydrolytic enzymes are now known to differentially mediate the progression of flower senescence. The individual importance of each signal appears to be species-specific and, in some instances, variety-specific, and varies differentially between floral organs. The challenge for postharvest scientists is to identify a hierarchy of regulators or a specific pattern of events that progresses senescence for certain groups of flower species. Subsequent categorization of cut flowers based on their metabolism and sensitivities will enable targeted application of appropriate postharvest technologies.

While differential cDNA screening, differential

display and cDNA subtraction have identified a number of senescence-related genes, the expression of most genes has not been investigated in flowers,

56 57

B. Ethylene biosynthesis, receptors and signal transduction

• More research is required to discover the developmental factors and regulatory genes that control the expression of ethylene biosynthesis genes.

• The only gene and enzyme that has not been fully understood in the ethylene biosynthesis pathway is related to the conversion of ACC into MACe. So, complete characterization of ACC­N malonyl transferase at gene and enzyme levels is important in view of its regulatory role in ethylene biosynthesis by diverting ACC from its normal route to ethylene.

• Identification of additional components involved in ethylene signal transduction, their J ur ther characterization and studies on the biochemistry of ripening are still essential for complete understanding of the climacteric ripening processes. This will also help us in understanding the process in vol ved in differential perception of ethylene during development as even today it is not well understood.

C. Climacteric and/versus non-climacteric ripening

• Recent work on the molecular basis of developmental ripening control suggested common regulators for climacteric and non­climacteric ripening. The work further indicated that the ripening of climacteric fruits comprises a portion of non-climacteric behaviour. So, an exhaustive identification of ethylene-dependent and ethylene-independent gene expression remains to be done. Further, little is known about the regulation of ripening in non-climacteric fruit and the upstream regulation of ethylene in climacteric fruits ..

• What is the exact reason and specific role of rise in respiration (climacteric rise) during ripening of climacteric fruits? During ripening of mango and tomato fruits alternate respiration (cyanide resistant respiration) is the major

portion of total respiration . Its role, significance and manipulation in slowing down the ripening process need the attention.

D. Storage

• Identification of threshold values of water loss that induce hormonal changes; various other physiological, biochemical and biophysical responses; noticeable shriveling an d physiological disorders .

• How to minimize the deterioration at quality levels during the prolonged storage of fruits (not sensitive to chilling injuries) at low temperature such as apple?

E. Physiological disorders

• Post-harvest disorders having phy siological reasons need to be studied in view of regulating the requirements which can decrease the development of such physiological disorders. Usually; temperature, a/co

2 ratio, rate of

ventilation, mineral content in tissue, pre-harvest factors, carbohydrate metabolism, and oxidative changes are involved for such disorders. Thus, understanding the physiological basis of such disorders will help in overcoming the losses by taking required pre-harvest and post-harvest precautions.

• Understanding the reasons for inter-cultivar differences in susceptibility to vari ous physiological disorders including chilling injury. Investigations on the physiological and biochemical basis of controlled atmosphere­induced physiological disorders and how these could be minimized during storage. Further, optimizing nutrition and endogenous hormonal balance to reduce the incidence and severity of various physiological disorders ne ed the attention.

F. Traditional approach, mutants and transgenic

• Development of rapid, reliable, repeatable and efficient regeneration and transformation protocols of fruit plants including the woody species.

• In future, technology or the availability of methodology would not be a limiting factor but, scope of genetically engineered fruits with suitable traits would more depend on economics and consumers' preference.

• It would be better that instead of interfering with whole of the ripening process, attention should be to target very specific proce sses such as, the fruit colour development or the kinetics of sugar accumulation in fruits.

• Private companies are too eager to cash in on the new technologies/transgenics and so many new products are being developed without a full understanding of the metabolic processes taking place especially at the levels of regulation and the direct, indirect and subsequent effects of introduced change. Thereby, we are running the risk of developing products with unintended but potentially adverse agronomic, adaptable and acclimatizing characteristics at whole plant or/ and crop levels.

• For economically backward and developing countries molecular and biotechnological research should not be at the expense of traditional and other innovative approaches of crop improvement and other already well established disciplines of agricultural sciences.

G. New and innovative ways and means to control ripening

• To discover, invent or synthesis the novel compounds preferably volatile but non-toxic in nature that could either inhibit or delay the ripening process when applied to pre or post harvested fruits.

• Ethanol is a promising, cost-effective and non­toxic chemical that can delays the ripening in tomato fruits . This therefore opened up the research to further standardize the dose of ethanol which can effectively delay the ripening irrespective of variety, for different durations and for fruits at different ripening stages so that the required basic information can be generated for the large scale application of ethanol to delay the ripening.

• Reproductive physiology in terms oftiming of floral induction, rate of flower development, extent of flowering, fruit growth, and developmental, incompatibility, sterility, parthenogenesis, parthenocarpy and apomixes (including the method of micropropagation) is not fully exploited at commercial scale in India for improving overall production of fruits , vegetable and flowers.

• Precise understanding of physiological basis for flower and pre-mature fruit drop will also contribute significantly towards enhancing the productivity and production.

IV. Future prospects for post-harvest physiology of flower senescence

There is little doubt that the molecular and genetic analyses of flower senescence made in the past 5 years have raised our awareness of the complex interactions that occur to regulate flower development and senescence. Genetic technologies have enabled scientists to search for senescence­related genes in plants often described as science models (e.g., Petunia, Arabidopsisi, and then translate the data into other species to determine the functional significance of the expression of specific genes in specific tissues after harvest. Interactions between ethylene, cytokinin, sugars and various hydrolytic enzymes are now known to differentially mediate the progression of flower senescence. The individual importance of each signal appears to be species-specific and, in some instances, variety-specific, and varies differentially between floral organs. The challenge for postharvest scientists is to identify a hierarchy of regulators or a specific pattern of events that progresses senescence for certain groups of flower species. Subsequent categorization of cut flowers based on their metabolism and sensitivities will enable targeted application of appropriate postharvest technologies.

While differential cDNA screening, differential

display and cDNA subtraction have identified a number of senescence-related genes, the expression of most genes has not been investigated in flowers,

56 57

leaves and fruits . The use of enhancer trap lines in Arabidopsis has resulted in the identification of over one hundred lines that have reporter gene expression in senescing but not in non-senescing ti ssues. This technology starts to reveal the complexity of the network of senescence-regulated pathways and will allow for the identification of many additional senescence related genes . The identification of se nes cence specific promoter elements and the generation of mutants and transgenic plants will help us to better understand the regulation of senescence related genes during senescence. DNA micro-arrays will allow temporal and spatial expression patterns. to be determined for hundreds of genes involved in senescence. These technologies will lead to an increased understanding of the initiation and execution of senescence which will allow us to increase vase lif~ and horticultural performance of ornamentals, increase yield in agronomic crops and decrease post-harvest losses of fruits and vegetables.

V. Conclusions

Our aims of pre serving the natural resources and to stop their over-exploitation can partly be achieved by reducing po st -harvest losses to a minimum level. This will make the availability of the basic commodities like fruit s, vegetables and flowers to growing population without further increasing the land under agricultural operations. Adoption of appropriate post-harvest management practices in terms of utilization, storage, processing etc . will not only reduce wastage of most of perishable fruits, vegetables and flowers but also help in managing gluts and price destabilization which adversely affect the farmers . To achieve this, understanding the physiological and molecular basis of fruit ripening and senescence would be helpful. Focused research with defined objectives and coherent attitude of different disciplines towards the de ve lop me n t of cost-effective, location-specific and env ironment-friendly pre and post-harvest technologies will bridge the existing gaps present in t-rms of inadequate availability, quantity and quality. Thi s would also ensure the proper transportation/distribution of harvested

commodity with respect to time and distance, gain of health for the consumers of fruits and economic incentives to fruit and tlower growers especially the poor farmers with limited resources .

SELECTED REFERENCES Arora, A (2005). Ethylene receptors and molecular

mechanism of ethylene sensitivity in plants . Curro Sci. 89 : 1348-1361.

Arora, A. (2007a) . Biochemistry of Flower Senescence. In: Paliyath, G , Murr, D.P., Handa, A.K., Lurie, S. (eds.): Postharvest Biology and Technology of Fruits, Vegetables and Flowers. Blackwell Publishing, Iowa, USA, (In pre ss).

Arora, A (2007b). Programmed Cell Death During Plant Senescence. In : Paliyath, G, Murr, D.P., Handa, A.K ., Lurie , S. (eds .) : Postharvest Biology and Technology of Fruits, Vegetables and Flowers. Blackwell Publishing, Iowa, USA, (In press) .

Arora, A and Ezura, H. (2003). Isolation, molecular characteri zation and regulation of cysteine protease gene in Gladiolus g randiflora . Molecular and Cellular Proteomics 2: 746.

Arora, A and Singh, VP. (2004). Cysteine protease gene expression and proteolytic activity during floral dev elopment and senescence in ethylene­insensitive gladiolus . Journal of Plan t Biochemistry and Biotechnology 13: 123-126.

Arora, A and Singh, VP. (2006) . Polyols regulate the flower senescence by delaying programmed cell death in Gla dio lus. Journal oj Plan t Biochemistry and Biotechnology 15: 139-142.

Arora, A. and Singh, VP. (2007). RNA interference: a novel approac h for gene silencing. In : Recent Advances in Plant Sciences. Edited by Setia, x.c., Setia, N. , Thind, S. K. and Nayyar, H. , Punjab Agricultural University, Ludhiana.

Arora, A., Sairam, R .K. and Srivastava, G.<::; . (2002) . Oxidative stress and antioxidativ e system in plants. Curro Sci . 82 : 1227-1238.

Arora, A , Singh, VP. , Sindhu , S.S., Rao, D.N. and Voleti , S .R. (2007) . Oxidative stres s mechanisms during fl ower senescence . In :

58

Floriculture , Ornamental and Plant Biotechnology: Advances and Topical Issues . (Ed. Jaime A Teixeira da Silva), Global Science Books, Ltd. , London, UK.

Arora, A, Watanabe, S., Ma, B., Takada, K. and Ezura, H. (2006) . A novel ethylene receptor homolog gene isolated from ethylene-insensitive flowers of gladiolus (Gladiolus grandiflora hort. ). Biochemical and Biophysical Research Communications Dec 22: 351 : 739-44. Epub 2006 Oct 30.

Elanchezhian, R. and Srivastava, G.c. (200 la). Effect of growth regulators on senescence of chrysanthemum flowers . Ind ian J . Plant Physiol. 6 NS : 233-243.

Elanchezhian, R. and Srivastava, GC. (2001b) . Physiological re sponses of chrysanthemum petals during senescence. Biol. Plant. 44: 411­415 .

Ezhilmathi, K. , Singh, VP. Arora, A and Sairam, R.K. (2007). Effect of 5-sulfosalicylic acid on antioxidant activity in relation to vase life of Gladiolus cut flowers. Plant Growth Regulation. 51: 99-108

Komal Mathur and Srivastava, GC. (2005). Effect of I-MCP on malic enzyme activity and ethylene production in mango during ripening. Indian J. Plant Physiol . 10: 273-275 .

Kumar, N. Srivastava, GC., Dixit, K., Mahajan A, and Madan Pal (2007). Role of carbohydrate in flower bud opening in rose (Ro sa hybrida L.). 1. Hort. Sci. & Biotech. 82 : 235-242.

Pandey, M ., Srivastava, G.c. and Prasad, N .K. (1998). Physiological changes associ ated with ripening of two mango varieties. Indian 1. Plant Physiol . 3: 94-96.

Pandey, M., Zeng Yanru , Prasad, N.K. and Srivastava, GC. (1995) . Alternate respiration during ripening of tomato fruits. Indian J. Plant Physiol. 38: 182-183.

Pau l, V and Srivastava, G C . (2006) . Role of surface morphology in determining the ripening behaviour of tomato (Lycopersicon esculentum

59

Mill.) fruits . Sci . Hort. 110: 84-92 .

Paul, V, Arora, A and Srivastava, G C. (2005) . Plant Senescence Process and Productivity. In : Plant Molecular Physiology, Trivedi, P. C. (ed.), Aavishkar Publisher, Jaipur, Rajasthan, India, pp . 215 -236.

Paul, V, Malik, S . K. and Srivastava, G C. (2007). Intervareital differences in the surface morphology and anatomy of mango (Mangifera indica L.) fruit s. Phytomorphology 57: (In Pre ss).

Paul, V, Srivastava, G C. and Singh, V P. (2005) . Changes in electrolyte efflux pattern in detached and attached tomato (Lycop ersicon esculentum Mill.) fruits in slow and fast ripening varieties. Indian J Plant Physiology 10: 25-31.

Prasad, N .K., Srivastava, G'C, and Pandey, M. (1999a). Studies on mango fruit ripening with reference to superoxide di smutase and polygactouronase enzyme activity under different storage conditions. 1. Plant Bio l. 26 : 161-164.

Prasad, N.K., Srivastava, Gc. and Pandey, M . (1999b). Effect of partial atmospheric pressure on th e she lf l ife of mango fruit s . Plant Physiology for sustainable Agriculture (ed . GC. Srivastava), Pointer Publication, pp. 273-281.

Pushpalatha, P., Singh, Atar and Srivastava, G'C. (2006). Effect of l-methylecyclopropene on ripening and associated parameters in tomato fruits. Indian J. Plant Physiol. 11: 234-238.

Reddy, YV and Srivastava, G.C. (1998). Malic enzyme and cyanide re si stant respiration in mango during ripening. Indian J. Plant Physiol. 3: 185-187.

Reddy, Y V and Srivastava, Gc. (l999a) . Ethylene biosynthesis and respiration in mango fruits during ripening. Indian J. Plant Physiol. 4: 32­35.

Reddy, Y.v. and Srivastava , o.c. (l999b) . Regulation of cell wall softening enzymes during ripening of mango fruits . Indian J. Plant Physiol. 4: 194-196.

leaves and fruits . The use of enhancer trap lines in Arabidopsis has resulted in the identification of over one hundred lines that have reporter gene expression in senescing but not in non-senescing ti ssues. This technology starts to reveal the complexity of the network of senescence-regulated pathways and will allow for the identification of many additional senescence related genes . The identification of se nes cence specific promoter elements and the generation of mutants and transgenic plants will help us to better understand the regulation of senescence related genes during senescence. DNA micro-arrays will allow temporal and spatial expression patterns. to be determined for hundreds of genes involved in senescence. These technologies will lead to an increased understanding of the initiation and execution of senescence which will allow us to increase vase lif~ and horticultural performance of ornamentals, increase yield in agronomic crops and decrease post-harvest losses of fruits and vegetables.

V. Conclusions

Our aims of pre serving the natural resources and to stop their over-exploitation can partly be achieved by reducing po st -harvest losses to a minimum level. This will make the availability of the basic commodities like fruit s, vegetables and flowers to growing population without further increasing the land under agricultural operations. Adoption of appropriate post-harvest management practices in terms of utilization, storage, processing etc . will not only reduce wastage of most of perishable fruits, vegetables and flowers but also help in managing gluts and price destabilization which adversely affect the farmers . To achieve this, understanding the physiological and molecular basis of fruit ripening and senescence would be helpful. Focused research with defined objectives and coherent attitude of different disciplines towards the de ve lop me n t of cost-effective, location-specific and env ironment-friendly pre and post-harvest technologies will bridge the existing gaps present in t-rms of inadequate availability, quantity and quality. Thi s would also ensure the proper transportation/distribution of harvested

commodity with respect to time and distance, gain of health for the consumers of fruits and economic incentives to fruit and tlower growers especially the poor farmers with limited resources .

SELECTED REFERENCES Arora, A (2005). Ethylene receptors and molecular

mechanism of ethylene sensitivity in plants . Curro Sci. 89 : 1348-1361.

Arora, A. (2007a) . Biochemistry of Flower Senescence. In: Paliyath, G , Murr, D.P., Handa, A.K., Lurie, S. (eds.): Postharvest Biology and Technology of Fruits, Vegetables and Flowers. Blackwell Publishing, Iowa, USA, (In pre ss).

Arora, A (2007b). Programmed Cell Death During Plant Senescence. In : Paliyath, G, Murr, D.P., Handa, A.K ., Lurie , S. (eds .) : Postharvest Biology and Technology of Fruits, Vegetables and Flowers. Blackwell Publishing, Iowa, USA, (In press) .

Arora, A and Ezura, H. (2003). Isolation, molecular characteri zation and regulation of cysteine protease gene in Gladiolus g randiflora . Molecular and Cellular Proteomics 2: 746.

Arora, A and Singh, VP. (2004). Cysteine protease gene expression and proteolytic activity during floral dev elopment and senescence in ethylene­insensitive gladiolus . Journal of Plan t Biochemistry and Biotechnology 13: 123-126.

Arora, A and Singh, VP. (2006) . Polyols regulate the flower senescence by delaying programmed cell death in Gla dio lus. Journal oj Plan t Biochemistry and Biotechnology 15: 139-142.

Arora, A. and Singh, VP. (2007). RNA interference: a novel approac h for gene silencing. In : Recent Advances in Plant Sciences. Edited by Setia, x.c., Setia, N. , Thind, S. K. and Nayyar, H. , Punjab Agricultural University, Ludhiana.

Arora, A., Sairam, R .K. and Srivastava, G.<::; . (2002) . Oxidative stress and antioxidativ e system in plants. Curro Sci . 82 : 1227-1238.

Arora, A , Singh, VP. , Sindhu , S.S., Rao, D.N. and Voleti , S .R. (2007) . Oxidative stres s mechanisms during fl ower senescence . In :

58

Floriculture , Ornamental and Plant Biotechnology: Advances and Topical Issues . (Ed. Jaime A Teixeira da Silva), Global Science Books, Ltd. , London, UK.

Arora, A, Watanabe, S., Ma, B., Takada, K. and Ezura, H. (2006) . A novel ethylene receptor homolog gene isolated from ethylene-insensitive flowers of gladiolus (Gladiolus grandiflora hort. ). Biochemical and Biophysical Research Communications Dec 22: 351 : 739-44. Epub 2006 Oct 30.

Elanchezhian, R. and Srivastava, G.c. (200 la). Effect of growth regulators on senescence of chrysanthemum flowers . Ind ian J . Plant Physiol. 6 NS : 233-243.

Elanchezhian, R. and Srivastava, GC. (2001b) . Physiological re sponses of chrysanthemum petals during senescence. Biol. Plant. 44: 411­415 .

Ezhilmathi, K. , Singh, VP. Arora, A and Sairam, R.K. (2007). Effect of 5-sulfosalicylic acid on antioxidant activity in relation to vase life of Gladiolus cut flowers. Plant Growth Regulation. 51: 99-108

Komal Mathur and Srivastava, GC. (2005). Effect of I-MCP on malic enzyme activity and ethylene production in mango during ripening. Indian J. Plant Physiol . 10: 273-275 .

Kumar, N. Srivastava, GC., Dixit, K., Mahajan A, and Madan Pal (2007). Role of carbohydrate in flower bud opening in rose (Ro sa hybrida L.). 1. Hort. Sci. & Biotech. 82 : 235-242.

Pandey, M ., Srivastava, G.c. and Prasad, N .K. (1998). Physiological changes associ ated with ripening of two mango varieties. Indian 1. Plant Physiol . 3: 94-96.

Pandey, M., Zeng Yanru , Prasad, N.K. and Srivastava, GC. (1995) . Alternate respiration during ripening of tomato fruits. Indian J. Plant Physiol. 38: 182-183.

Pau l, V and Srivastava, G C . (2006) . Role of surface morphology in determining the ripening behaviour of tomato (Lycopersicon esculentum

59

Mill.) fruits . Sci . Hort. 110: 84-92 .

Paul, V, Arora, A and Srivastava, G C. (2005) . Plant Senescence Process and Productivity. In : Plant Molecular Physiology, Trivedi, P. C. (ed.), Aavishkar Publisher, Jaipur, Rajasthan, India, pp . 215 -236.

Paul, V, Malik, S . K. and Srivastava, G C. (2007). Intervareital differences in the surface morphology and anatomy of mango (Mangifera indica L.) fruit s. Phytomorphology 57: (In Pre ss).

Paul, V, Srivastava, G C. and Singh, V P. (2005) . Changes in electrolyte efflux pattern in detached and attached tomato (Lycop ersicon esculentum Mill.) fruits in slow and fast ripening varieties. Indian J Plant Physiology 10: 25-31.

Prasad, N .K., Srivastava, G'C, and Pandey, M. (1999a). Studies on mango fruit ripening with reference to superoxide di smutase and polygactouronase enzyme activity under different storage conditions. 1. Plant Bio l. 26 : 161-164.

Prasad, N.K., Srivastava, Gc. and Pandey, M . (1999b). Effect of partial atmospheric pressure on th e she lf l ife of mango fruit s . Plant Physiology for sustainable Agriculture (ed . GC. Srivastava), Pointer Publication, pp. 273-281.

Pushpalatha, P., Singh, Atar and Srivastava, G'C. (2006). Effect of l-methylecyclopropene on ripening and associated parameters in tomato fruits. Indian J. Plant Physiol. 11: 234-238.

Reddy, YV and Srivastava, G.C. (1998). Malic enzyme and cyanide re si stant respiration in mango during ripening. Indian J. Plant Physiol. 3: 185-187.

Reddy, Y V and Srivastava, Gc. (l999a) . Ethylene biosynthesis and respiration in mango fruits during ripening. Indian J. Plant Physiol. 4: 32­35.

Reddy, Y.v. and Srivastava , o.c. (l999b) . Regulation of cell wall softening enzymes during ripening of mango fruits . Indian J. Plant Physiol. 4: 194-196.

Reddy, YY. and Srivastava, Gc. (2001). Ethylene biosynhesis and respiration during ripening of mango cultivars. Indian J. Plant Physioi. 6: 361­364.

Reddy, YV. and Srivastava, G.C . (2003). Superoxide dismutase and peroxidase activ ities in ripening mango (Mangifera indica L.) fruits. Indian J. Plant Physioi. 8: 115-119.

Sharma-Natu, Poonam, Ghildiyal, M .C . and Srivastava, G.C. (2003). Plant physiology research at IARI (Eds. Pant, R.C . and Ghildiyal, M .C .), pp . 146-152 . In: Souvenir : 2nd International Congress of Plant Physiology on 'Sustainable plant productivity under changing environment', Jan . 8-12, 2003 .

Singh, A., Kumar, 1., Kumar, P. and Singh, Y. P. • (2005a). Influence of 8-hydroxy quinoline (8­

HQ) and sucrose pulsing on membrane stability and post-harvest quality of gladiolus cut spikes. 1. Om. Hort. 8: 243-248.

Singh, V. P. (2005). Deterioration of membrane during flower senescence in gladiolus flowers and its amelioration with free radical scavenger. J. Om. Hort. 8: 8-12 .

Singh, V. P. and Jegadheesan, A. (2003). Effect of alpha-lipoic acid on senescence in gladiolus flowers. Indian 1. Plant Physii. (Spl Issue No.1):

72-79 .

Singh, v.P., Kiran, D. and Arora, A. (2005b) . Alleviation of antioxidants activity in gladiolus flowers during senescence by spermine and spermidine. 1. Om. Hort . 8: 167-172 .

Singh, v.P., Kiran, D. and Arora, A. (2005c) . Effect of spermine, spermidine and putrescine on the vase life and associated parameters in two Gladiolus varieties. 1. Om. Hort. 8: 161- 166.

Singh,Y.P and Srivastava,G.C. (2006) . Physiological and biochemical basis of flower senescence. In: Advances in Ornamental Horticulture, Vol. 5 (S.K. Bhattachrjee ed .), Pointer Publishers, pp . 105-119 .

Srivastava, GC. (1999) . Physiological and biochemical basis of ripening and senescence. In: Manual, Post Harvest Management of Fruits, Vegetables and Flowers. ICAR Sponsored Programme, pp. 30-33.

Srivastava, GC. (2003). Post -harvest physiology of cut flowers . Floriculture Today 8: 29-31.

Srivastava, G C. and Paul, V. (2003). Post-harvest technology of fresh fruits, vegetables and flowers : Role of Plant Physiology. In: Souvenir (Eds . Pant, R . C. and Ghildiyal, M. C.) 2nd

International Congress of Plant Physiology on "Sustainable Plant Productivity Under Changing Environment" held during 8-12 January, 2003, IARI, New Delhi 110 012, India, pp : 20-26.

Srivastava, GC., Zeng Yanru , Pandey, M and Prasad, N.K. (1996) . Effect of silver nitrate on activity of malic enzyme during ripening in mango (Mangifera indica) . Indian J. Exp. Bioi. 34 (June): 575-576.

Venu, P. and Srivastava, GC. (2000) . Rose flower senescence: Dealing an elephant in the dark. Physioi. Mol . Bioi. Plants 7: 3-5.

Zeng Yanru, Pandey, M. Prasad, N.K. an d Srivastava, Gc. (1995). Ripening associated changes in enzymes and respiratory activities in three varieties of mango (Mangifera indica L. ). Indian J. Plant Physioi. 38: 73-76.

Zeng Yanru, Pandey, M., Prasad, N .K. an d Srivastava, Gc. (1996). Hydrolysing enzymes and respiration during ripening of tomato (Lycope rsicon esculentum) fruits. Curro Sci . 70:1017-1018.

CROP RESPONSES TO CLIMATE CHANGE Rakesh Pandey and M.e. Ghildiyal

Division of Plant Physiology

Indian Agricultural Research Institute , New Delhi 110 012

INTRODUCTION The earth climate is showing an alarming rise

in the concentration of greenhouse gases consequently, an increase in temperature is projected. There is an increase in the incidence and intensity of floods, droughts, hot spells and land degradation which are a serious threat to the agriculture and livelihood of farming community. Several anthropogenic factors such as deforestation, urbanization, industrialization, pollutants and loss of ozone layer have contributed to the pace of recent climate change. There is a description of the effect of human actions on the climate and agriculture in ShimadBhagvadgita (Chapter 3, verse 14) as follows ­

3i?l 1@Cl F~ ~ q4 '::lllC'Wi'qCf: I

<r~~ ~: Cfi4+i:J)@Cl: 11

All creatures develop from food (grains) and rains make possible the grains.

The rains are due to yajya and yajya is created by actions.

The United Nations cl imate agency - Inter Governmental Panel on Climate Change (lPCC) has created an ever broader informed consensus about connection between human activities and global warming. The 2007 Nobel Peace Prize has been recently awarded to Al Gore and the IPCC for their efforts to build up and disseminate knowledge about man made climate change and to lay foundation for the measure that are needed to counteract such changes.

IPCC (2007) has projected that the global warming would raise average temperature of the earth surface by 2.0 to 4.0 DC by the end of this century. The carbon dioxide concentration in the

atmosphere will also rise to 570 imol mo l:' by the middle of this century. Although higher CO will

2

benefit especially the C crops due to higher3

photosynthesis but other crops (C4

type) may not benefit. The rising temperature and water scarcity may also nullify or reduce the beneficial effects of CO

2 on crop plants. The increase in UV-B radiation

will also affect the crop growth and yield .

In order to understand the crop responses to climate change, work on different aspects was initiated in the Division of Plant Physiology at IARI, New Delhi . The objectives of these studies and the ongoing work are as follows ­

1. To develop facilities for CO2enrichment, UY­

B radiation and temperature interaction studies.

2. To study the morpho-physiological and biochemical responses of crop plants to elevated CO UV-B radiation and interactive

2,

effects of CO and temperature. 2

3. Identification of crops/genotypes and crop management practices for alleviating the effects of climate change.

4. Application of crop response data for modeling and developing plant types.

EXPERIM ENTAL SET- UP AN D FACILITIES The most important designs and facilities used

in elevated CO 2 studies are - open top chambers (OTC) and free air CO enrichment (FACE)

2

technology. Most of the studies have been done using OTCs in whic h CO

2 enrichment is done in

partially enclosed conditions. The OTCs can also be used for CO 2and temperature interaction studies . However, FACE grown crops are enriched with CO

2 in open and have a natural crop microclimate.

60 61