Biochemical changes in the composition of developing seeds of Pongamia pinnata (L.) Pierre

Industrial Crops and Products 53 (2014) 199–208

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Biochemical changes in the composition of developing seeds of Pongamia pinnata (L.) Pierre H.R. Pavithra a,b , Balakrishna Gowda a,∗ , M.B. Shivanna b a b

Biofuel Park, Department of Forestry and Environmental Science, University of Agricultural Sciences, GKVK, Bengaluru 560065, Karnataka, India Department of Applied Botany, Kuvempu University, Jnana Sahyadri, Shankaraghatta, Shimoga 577451, Karnataka, India

a r t i c l e

i n f o

Article history: Received 2 June 2013 Received in revised form 13 December 2013 Accepted 20 December 2013 Keywords: Pongamia pinnata Seed development Reserve material Mineral nutrients Amino acids

a b s t r a c t The biochemical changes occurring in developing Pongamia pinnata seeds were determined at an interval of three weeks from 30 weeks up to 42 weeks after flowering. Significant variation in total sugar, starch, lipid, protein and oil body associated protein contents was documented. The total carbon content decreased significantly, while nitrogen content increased. Significant variation in mineral nutrient content was also detected across all the stages of seed development. Oil body associated protein-specific band between 20 and 19 kDa was prominently observed at later stages of seed development. Phytic acid content increased from 0.58 to 2.35%. Steady decrease in chlorophyll content from 0.175 to 0.013 mg g−1 of seed dry wt. was observed. Electrical conductivity decreased during the seed development. The crude fibre content increased, while the ash content remained constant at all stages of seed maturity. Quantitative changes in amino acids during seed development were observed. Seeds harvested at 42 weeks after flowering had maximum physiological maturity with high oil content and seed reserve material. The base line data of pongamia seed development could be used in the furtherance of knowledge relating to molecular, physiological and genetic aspects regulating biosynthetic pathways of reserve materials. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Pongamia pinnata (L.) Pierre indigenous to Indian sub-continent [family: Fabaceae; Syn. Pongamia glabra Vent., Derris indica (Lam.) Bennett and Millettia pinnata (L.) Panigrahi] is a multipurpose tree species with extensive nitrogen fixing ability and used for medicine, fuel, manure, fodder and as insecticide (Shivanna and Rajakumar, 2010; Pavela, 2009). The pongamia oil is biodegradable and hence identified as one of the best alternatives to fossil fuels (Naik et al., 2008). The species is being cultivated in the United States of America, Australia and China for its multiple uses (Anon., 1969). Pongamia oil is also known for karanjin, karanjone and diketone pongamol (Yadav et al., 2004). The tree requires 6–7 year for fruiting and pods are harvested during February–April. In order to achieve maximum seed production and tree improvement, extensive studies on physiological and biochemical changes during seed maturation have been carried out. Proper seed development is crucial for plant survival, its productivity and economical importance (Suda and Giorgini, 2000).

Abbreviations: WAF, weeks after flowering; PSV, protein storage vacuoles. ∗ Corresponding author. Tel.: +91 80 23620022; fax: +91 80 23330277. E-mail address: [email protected] (B. Gowda). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.12.032

In a legume seed, reserve biochemical compounds like lipids, proteins and carbohydrates accumulate gradually during seed maturation (Djemel et al., 2005). The carbon, nitrogen and amino acid partitioning between oil, carbohydrates and proteins in developing seeds have been well understood (Ekman et al., 2008; Schiltz et al., 2005). The mineral nutrients that constitute less than 3% of the seed dry mass form an important source of essential nutrients. In legumes like Pisum sativum, the accumulation of minerals is well documented (Grusak, 1994). In most legume seeds, certain antinutritional compounds are associated with seed storage protein, which limits the economic importance of these proteins (El-Niely, 2007). In view of this, there is a strong interest in the identification of the rate of accumulation of anti-nutritional compounds along with proteins during seed filling. The import of monomers like sugars and amino acids, certain mineral ions and macromolecules through sieve elements into the developing seeds is also reported (Weber et al., 1997). In the previous study, the histological and ultrastructural evidence for the accumulation of reserve material during seed development was recorded (Pavithra et al., 2013). The ideal time for harvesting of mature fruits was determined at 42 weeks after flowering (WAF) when fruits accumulated maximum contents of oil, oleic acid and karanjin (Pavithra et al., 2012). However, no information is available regarding the biochemical changes occurring during different developmental stages of pongamia seeds. In

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this context, experiments were designed to profile the biochemical changes that occur during the development of pongamia seeds. 2. Materials and methods 2.1. Identification of trees for collection of seed samples Ten pongamia trees (20-year-old) at flowering stage in the campus of Gandhi Krishi Vignana Kendra, University of Agricultural Science, Bengaluru, India were identified and marked for the collection of seed samples. The essential characteristics of trees include five to ten sub-branches, circular canopy with drooping branches and heavy pod yield. The inflorescences were identified a few days before the anthesis of flowers (unfolding of standard petal indicated flower opening; Raju and Rao, 2006) during March–May. Throughout the duration of flowering, the inflorescences were monitored and on the third day closed flowers without corolla (indicating completion of pollination) were tagged. The tagged pods of all the marked trees were harvested at an interval of three weeks starting from 30 WAF until 42 WAF and pods at each specific developmental stage were pooled separately. The pods (both single and two seeded) were studied for their morphological characteristics. The pod samples of specific stages were stored in sealed ziplock plastic bags at −20 ◦ C. The seeds were separated manually from pods and studied for the following biochemical characteristics with three replications for each analysis. 2.2. Determination of total sugar and starch content The total sugar content was determined by the method described by Siloto et al. (2006) with some modifications. Accordingly, 0.5 g of seed powder was homogenized in 80% (v/v) ethanol and incubated at 70 ◦ C for 90 min. The homogenate was centrifuged at 10,820 × g for 5 min and the supernatant was collected. The pellet was washed three times with 0.5 ml of 80% ethanol and all supernatants were combined from each wash and evaporated at room temp. The resultant residue which represented the total sugar was dissolved in 0.5 ml of water and estimated by phenol-sulphuric acid method. The insoluble fraction or the pellet from the ethanol extraction was suspended in 0.4 ml of 0.2 M KOH and incubated at 95 ◦ C for 1 h. The solution was neutralized with 75 ␮l of 1 M acetic acid and centrifuged at 10,820 × g for 5 min. The supernatant was used for quantification of starch by anthrone method (Hodge and Hofreiter, 1962). 2.3. Determination of total lipid content The total lipids were extracted according to the method described by Bligh and Dyer (1959) and were quantified gravimetrically after drying in a desiccator at 24 ◦ C for 4 h.

acetone was added to 100 ␮l of supernatant and incubated at 4 ◦ C overnight. Samples were centrifuged at 15,652 × g at 4 ◦ C for 15 min to pellet the protein. Then protein pellet was dissolved in 100 ␮l of distilled water. The protein content of the extract was determined by the method of Lowry et al. (1951). The crude protein content was calculated by multiplying the total nitrogen content with 6.25. 2.6. Determination of oil-body associated protein Oil bodies were isolated by flotation centrifugation method (Van Rooijen and Moloney, 1995) with minor modification. The seed samples were ground in 5 ml of oil body extraction buffer (0.4 M sucrose, 0.5 M NaCl, 0.05 M Tris HCl, pH 8.0) and centrifuged at 10,820 × g at 4 ◦ C for 15 min. The oil bodies were washed once in high stringency buffer (8 M urea, 0.1 M Na2 CO3 , pH 10) and centrifuged at 10,820 × g at 4 ◦ C for 10 min. The oil bodies were then washed twice with water. The oil body associated protein was extracted according to the method described by Katavic et al. (2006). The protein content of the extract was determined by Lowry’s method as described previously. 2.7. Determination of micro and macro-nutrients The seed sample (0.5 g) was digested with 8 ml of conc. nitric acid. The sample was incubated at 90 ◦ C for 30 min on a waterbath. Samples were cooled and 2 ml of hydrogen peroxide was added. The solution was heated on a hot plate until the sample volume is reduced to half. Then the solution was made up to 50 ml with distilled water. Potassium was measured with a Flame Atomic Emission Spectrophotometer (CL411, Chemiline, India). Zinc, copper, iron, manganese and potassium contents were measured with Atomic Absorption Spectrometer (AS 800, Perkin Elmer, USA). The total phosphorus content in seed sample was determined spectrophotometrically following nitric acid and hydrogen peroxide digestion using meta-vanadate method, as described by Bernhardt and Wreath (1955). 2.8. Determination of phytic acid The phytic acid in seed sample was determined according to the method described by Wheeler and Ferrel (1971) using ferric nitrate as the standard. The phytate phosphorus was calculated from the ferric ion concentration assuming 4:6 (Fe:P) molar ratio. 2.9. Determination of crude fibre and ash content The seed samples at different stages of maturity were determined for crude fibre and ash content according to the AOAC method (Anon., 1990).

2.4. Determination of total carbon and nitrogen content

2.10. Determination of electrical conductivity

The seed powder (0.5 g) was dried at 80 ◦ C to a constant wt. in a hot-air oven. The carbon and nitrogen content was quantified using the Isotope Ratio Mass spectrometer (IRMS, Thermo Finnigan, Germany).

Ten seeds in three replications were surface disinfected with mercuric chloride (1% for 2 min) solution and washed thrice with distilled water. Seeds were soaked in 50 ml distilled water and kept at room temperature for 18 h. The electric conductivity of seed leachate was measured using a digital conductivity metre (D1909/DS 7007, Systronics, India).

2.5. Determination of total soluble protein The seed powder was defatted using a mixture of chloroform:methanol:acetone (2:1:1) and 0.5 g of defatted sample was ground in 0.125 M Tris (pH 6.8) buffer using chilled mortar and pestle and incubated at 4 ◦ C overnight. Samples were centrifuged at 15,652 × g at 4 ◦ C for 15 min. The supernatant was separated and the extraction was repeated for complete extraction. Chilled

2.11. Determination of chlorophyll content The seed powder (0.5 g) was extracted with N,N-dimethyl formamide as described by Morgan and Porath (1980). Absorbance was read at 645 and 663 nm using the spectrophotometer (Carry 60, Agilent, Germany).

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2.12. Amino acid profiling by HPLC The seed powder (0.5 g) was extracted in 4 ml of 4 N methane sulfonic acid. Vials were sealed and contents were digested at 110 ◦ C for 24 h. The contents were neutralized with 4 ml of 4 N sodium hydroxide. Samples were filtered and the filtrate was used for analysis. The samples were pre-column derivatized with ophthalaldehyde (25 mg OPA, 0.5 ml of methanol, 4.5 ml of 0.1 M borate buffer solution and 25 ␮l of beta-mercaptoethanol) (Sigma Aldrich, India). The reaction mixture was prepared by adding 10 ␮l of 0.1 M borate buffer, 20 ␮l of sample and 2 ␮l of OPA reagent and incubated for 3 min at room temp. with continuous vortexing and then 128 ␮l of water was added and vortexed for 1 min and 1 ␮l of this reaction mixture was loaded and separated on a Shimadzu HPLC (Japan) instrument with an SPD-M20A PDA detector. The analysis was carried out using a Zorbax Eclipse AAA 4.5 mm × 150 mm, 5 ␮m column. The flow rate was 0.64 ml min−1 and oven temp. was set at 40 ◦ C. The mobile phase consisted of A. 0.04 M potassium phosphate buffer (pH 6.9), B. ACN:MeOH:H2 O (45:45:10). An optimized step-wise gradient ranging from 2% of B for 0 to 0.01 min, 2% of B for 0.01 to 0.50 min, 57% of B for 0.50 to 30.00 min, 95% of B for 30.00 to 30.10 min, 95% of B for 30.10 to 33.00 min, 2% of B for 33.00 min, 2% of B for 33.00 to 33.10 min, 100% of B for 33.10 to 38.00 min, 100% of B for 38.00 to 44.00 min, 2% of B for 44.00 to 45.50 min, 2% of B for 45.50 to 46.55 min and 100% of A for 46.55 to 47.00 min. The amino acids were detected at 338 nm. Samples were analyzed by comparing the retention time using amino acid standard solution (Sigma Aldrich, India) and quantification was performed by area normalization method. 2.13. Statistical analysis Statistical analysis was done for the data generated using the standard methodology. Analysis of variance was computed following the procedure given by Panse and Sukhatme (1976) using the software MSTAT-c (ver. 5.1). Mean values were compared by Duncan’s multiple range test. 3. Results and discussion Pongamia trees started flowering during March–May in the study area. The fruit setting that was initiated during June–July was completed during January–February. Pods were flat initially but were bulged and fully formed by August–September with highly immature embryo. The embryo started developing from 30 WAF onwards during October until it attained physiological maturity. Three phases of pod development were identified – early green immature pod stage, half brown pod stage and late dark brown pod stage (Pavithra et al., 2012). The previous study on pongamia indicated that the pod length and pod breadth did not vary much, however the pod thickness varied with gradual increase in seed length, seed breadth and seed thickness up to 42 WAF. The oil content increased with seed maturity and the oleic acid content remained high at the end of fruit maturity, while the karanjin content varied significantly across different stages of pongamia seed development (Pavithra et al., 2012). 3.1. Carbohydrate and lipid content The seed development is accompanied by cell division, followed by cell differentiation, accumulation of reserve material and maturation. In order to determine the contribution of carbohydrates towards lipid accumulation in pongamia, the seed samples were analyzed for soluble sugars and starch. At early stages of seed filling, a considerable amount of high total soluble sugar content was detected in the seed. The total sugar content of seeds at

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30–42 WAF decreased significantly (P < 0.01) (Fig. 1) from 22.54 to 18.49% with increase in maturity. The gradual decrease in soluble sugar has also been reported in Zea mays (Cao et al., 2008), Glycine max (Sharma et al., 2011) and Brassica napus (Morley-Smith et al., 2008). Weber et al. (2005) opined that seed coat plays an important role in the metabolic control of seed development. The previous report on histochemical staining of seed coat of developing pongamia (30–42 WAF) showed positive result for sugars indicating an active sugar metabolism in the filling seeds (Pavithra et al., 2013). In legume seeds, the activity of cell wall bound invertase is very low in the cotyledon and high in the seed coat leading to the assimilation of soluble sugars in cotyledons at early stages of development. The steady decrease in total sugar content from 33 to 42 WAF could be due to the inter conversion of different classes of sugar. Generally, monosaccharides are high during the initial stages of development, while oligosaccharides are high at later stages of development impairing membrane fusion and increase seed longevity thereby promoting desiccation tolerance (Blackman et al., 1992). A significant (P < 0.01) increase in the total starch content was observed in pongamia seeds at different stages of development (Fig. 1). During the early stages of seed development up to 33 WAF, the starch content was 5.5% and afterwards, the starch content increased to 7.4% at 42 WAF. The starch content at 42 WAF (maturity) was higher than that reported by Sangwan et al. (2010) in pongamia. The above observation is contrary to that in developing Arabidopsis seed with starch content falling sharply at late maturation stage (Andriotis et al., 2010). The simultaneous increase in starch content and decrease in total soluble sugar suggested the role of starch as carbon reserve for lipid or oil synthesis from 36 to 42 WAF. In general starch synthesis during early stages of seed development was accompanied by accumulation of photosynthetic proteins which help in recycling the CO2 released during fatty acid synthesis as a source of carbon which further furnish the oil biosynthesis during later stages of seed development (King et al., 1997). This recycling of CO2 process in the oilseeds may contribute to the oil yield efficiency. This coincides with the high amount of chlorophyll content of developing seeds at 33 and 36 WAF in pongamia. Starch synthesis takes place in the developing plastids. The densely stained starch granules in the plastids at the later stages of seed development as revealed by the ultrastructure in pongamia could be related to the increase in starch content of seeds (Pavithra et al., 2013). The total lipid content of seeds at 30–42 WAF increased significantly (P < 0.01) (Fig. 1). During 30 and 33 WAF, the total lipid content was 47.12 and 49.28% respectively. Later, the total lipid content increased to 54.04% at 42 WAF. Since pongamia is an oilseed species, it accumulates large quantities of total lipids during seed development. The total lipid content was seven times more than the starch content at 42 WAF. This coincided with the maximum number of oil bodies per cell produced in pongamia at 39 and 42 WAF (Pavithra et al., 2013). The variation in fatty acid content during pongamia seed development stages has been documented (Pavithra et al., 2012). This pattern was similar to significant increase in total lipid content reported in Lesquerella fendleri (Chen et al., 2009) and Helianthus annuus seed development (Kaushik et al., 2010). However, Sharma et al. (2011) reported different pattern of total lipid accumulation in Glycine max with initially very low accumulation to significant increase at later stages of development. It has been reported earlier, that during fatty acid synthesis, pyruvate and glucose-6-phosphate are precursors in the initial step of seed biosynthetic pathway in plastids (Kang and Rawsthorne, 1994). It is at this point of time steady decrease in sugar content and increase in lipid content during the later stages of pongamia seed development was observed.

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Fig. 1. Accumulation of (a) total sugar, (b) total starch and (c) total lipid in seeds at different stages of fruit development in Pongamia pinnata (SED, standard errors of difference; CD, critical difference).

3.2. Protein content A significant variation in total crude (P < 0.05) and soluble protein content (P < 0.01) and oil body associated protein content (P < 0.01) of seeds was observed across different developmental stages (Fig. 2). The total crude protein content was 23.72% (30–33 WAF) which increased quickly thereafter to 28.33% at 42 WAF. The total soluble protein content was very low during 30–33 WAF which sharply increased to 19.77% during 36–42 WAF. The variation in oil body associated protein content was similar to the total soluble protein content (4.71–9.93%). To determine changes in storage protein profile associated with seed development, a temporal pattern of protein accumulation using SDS-PAGE has been studied (Fig. 3). Protein bands of low Mr (22–14.3 kDa) predominated at all stages of seed development, while high Mr protein bands (43–68 kDa) appeared only at the later stages (39 and 42 WAF). A protein subunit band appeared between 29 and 20 kDa which increased in intensity and thickness at later stages of seed development (39 and 42 WAF). The protein subunit migrating between 20 and 19 kDa increased at later stages of seed development. This band corresponded to oleosin protein subunit as reported in other oilseeds (Frandsen et al., 2001; Katavic et al., 2006). The increase in band intensity of oleosin corresponded with the increase in oil body associated protein content at later stages of seed development (39 and 42 WAF). The protein subunit band between 20 and 19 kDa corresponds with the increase in localization of oil bodies at later stages of seed development (Pavithra et al., 2013). The protein bands occurring at different developmental stages of pongamia seed concurred with that reported in seeds from Guwahati (Kesari and Rangan, 2011) with few differences in banding pattern between 29 and 20 kDa. The change in banding pattern could be attributed to the variation in the geographical location (Pavithra et al., 2010). Low Mr protein bands at the later stage of seed development coincided with that reported in Olea europaea (Wang et al., 2001) and L. fendleri (Chen et al., 2009). The banding pattern at different developmental stages coincided with the total protein content. In general, the total protein content in legume seeds is inclusive of storage proteins, house-keeping proteins,

biologically active enzyme, protease inhibitors, lectins and allergens (Gallardo et al., 2008). The major storage proteins in legume seeds are globulins which are composed of legumins and vicilins; these proteins have no reported catalytic activity. The above oil body associated protein might have a role in preventing the coalescence of mature oil bodies besides protecting storage oil body from hydrolytic enzymes during seed desiccation or germination. There appears to be no known common regulation of synthesis of storage protein and 19 kDa oil body associated protein since seed-specific genes might be sequentially activated at different development stages (Frandsen et al., 2001). 3.3. Micro, macro-nutrient and phytic acid content Significant variation in total C content (P < 0.01) of seeds was observed across different developmental stages (Fig. 4). The total C content decreased from 23.05 to 19.99%. The storage forms of carbon in seeds are starch, protein and oil which are economically important products for food, feed and industrial applications. Fatty acid synthesis in the developing oilseeds occurs predominantly in plastids and requires C in the form of acetyl Co-A units. The simultaneous synthesis of oil and storage protein results in competition for carbon during the seed development. The invertase and hexokinase catabolized reactions are the possible pathways by which C from sucrose could enter the storage metabolic pathway in seeds (Tomlinson et al., 2004). The increase in C content at 36 and 39 WAF also coincided with higher accumulation of total lipids in the developing seeds of pongamia. Generally in oilseeds, one third of the C in sucrose is lost by decarboxylation in fatty acid synthesis (Alonso et al., 2007; Schwender and Ohlrogge, 2002). The oilseeds refix much of the C for lipid biosynthesis during the green phase of seed development (Ruuska et al., 2004). The total N content of seeds varied (P < 0.05) from 3.79 to 4.53% during 30–42 WAF (Fig. 4). The total N content in seeds at 30 and 33 WAF did not change significantly whereas it increased at 36 WAF to 4.47% and remained constant until harvest. In legumes, N is supplied to the developing seed through phloem in the form of amino acids or amides for the synthesis of storage proteins

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Fig. 2. Accumulation of (d) total crude protein, (e) total soluble protein and (f) oil body protein in seeds at different stages of fruit development in Pongamia pinnata (SED, standard errors of difference; CD, critical difference).

(Weber et al., 2005). Studies related to uptake, assimilation and distribution of N in plants require in-depth study as they are involved in seed protein performance. An increase in total N coincided with an increase in seed dry biomass at later stages of development. As the N content and dry wt. accumulation increases, there is a parallel decrease in the relative moisture content of seed (Pavithra et al., 2012). Similar conclusions have been made in other legume seeds (Golombek et al., 2001; Schiltz et al., 2005). The N accumulation in seeds during filling might depend upon the soil mineral N supply, symbiotic fixation of atmospheric N or endogenous N accumulated in the vegetative parts (Schiltz et al., 2005). Significant variation (P < 0.01) in micronutrient and macronutrients like Zn, Cu, Fe, Mn and K contents of seeds was observed across different developmental stages (Fig. 4). The Zn content of the seed increased from 0.39 to 0.62 mg kg−1 of seed dry biomass until 39 WAF which further decreased to 0.57 mg kg−1 at maturity. Zinc accumulation was found to be high during the later stage of seed development (39 WAF) and this could be related to the protein content at 39 WAF. These results were in accordance with the increase in zinc content at the later stages of seed development in Triticum aestivum (Ozturk et al., 2006). Many enzymes require Zn for their integrity and function. It is the most important nutrient affecting protein synthesis in plants. The Cu content in the developing seed of pongamia was low at early stages (0.17 mg kg−1 ) but increased at 33 WAF and again decreased during 36–42 WAF to 0.18 mg kg−1 of seed dry biomass. These results were in accordance with the increase in Cu content at the mid stage of Oryza sativa seed development and decreasing at later stages (Iwai et al., 2012). Copper is mainly accumulated in plastids and low concentration of copper affects many physiological processes like photosynthesis, respiration, cell wall metabolism and nitrogen fixation (Gao et al., 2008). The Fe content in the developing seed of pongamia was low during the initial stages and increased at 33 WAF to 3.01 mg kg−1 of seed dry biomass and decreased during 36–42 WAF to 2.40 mg kg−1 of seed dry biomass. Fe is an important co-factor for many of the redox reactions involved in photosynthesis and respiration. Conte and Walker (2011) reported that Fe must be either bound to storage

protein or embedded in vacuoles to prevent cellular damage. Since Fe is an important component in chlorophyll in plants, both the Fe and chlorophyll contents roughly parallel at 33 WAF and 36 WAF and then drop at the final stages of seed development. Such a sharp decrease in iron and chlorophyll contents was reported in Glycine max (Laszlo, 1991). Manganese remobilization from vegetative structures to seed was observed during 30–42 WAF. The Mn content was low initially and increased at 33 WAF to 0.47 mg kg−1 of seed dry biomass and decreased during 36–42 WAF to 0.29 mg kg−1 of seed dry biomass. The protein storage vacuoles (PSV) in the developing cotyledon cell store Mn and Zn as phytic acid salts; they are also stored in endoplasmic reticulum. The Mn in PS II complex of photosynthetic electron transport chain coincides with high chlorophyll content of developing seeds. Such a variation in Mn content was also reported in Arabidopsis thaliana (Otegui et al., 2002). Potassium is translocated from the maternal tissue to embryo through phloem and mostly follows the path of C and N. The K content remained almost constant throughout the development of seed (4.13–4.82 mg kg−1 of seed dry biomass). The above results corroborated with that in Glycine max (Laszlo, 1994) and Vicia sativa (Samarah and Ereifej, 2009). The K ions are involved in cell turgor pressure and in sucrose unloading from seed coat to the developing embryo. The above findings suggest that the accumulation of macro and micronutrients from the vegetative tissue to developing seeds varies depending upon the elements. The total P content of seeds collected during 30–42 WAF decreased significantly (P < 0.01) from 3750 to 2170 mg kg−1 of seed dry biomass (Fig. 4), whereas phytic acid increased significantly (P < 0.01) from 0.58 to 2.35% (Fig. 5). At 33 and 36 WAF phytic acid accumulation was initiated which increased until maturity. The phytic acid content at maturity was much higher in pongamia than in other legumes like Lupinus albus (Mohamed and RayasDuarte, 1995). At the initial stages of seed development, P content of seeds was very high, while phytic acid content was low. Usually protein bodies are storage organelles of phytic acid in the form of crystal complexes of minerals (Mn, Zn, Fe) (Otegui et al., 2002; Welch, 1997). It has been reported previously that during

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Fig. 3. Total seed protein profiling of developing seeds of Pongamia pinnata. (MW, molecular weight: 10–100 kDa, M, marker: Lane 1–5 corresponds to 30, 33, 36, 39 and 42 WAF, respectively)

phytic acid biosynthesis, variation in the availability of inorganic P, myo-inositol and mineral ions might result in the variation in accumulation of phytate at maturity (Raboy, 2003). Vadivel and Biesalski (2012) reported slightly high phytic acid content in pongamia at the time of harvest than the proximate value. Such a variation in phytic acid content could be attributed to the influence of geographical location. Of late, phytic acid is increasingly used to treat kidney stone, diabetes, atherosclerosis, coronary heart disease and a variety of cancer (Greiner et al., 2006). 3.4. Chlorophyll content and electrical conductivity Significant variation in chlorophyll content (P < 0.01) of seeds was observed across different developmental stages (Fig. 5). The chlorophyll content was 0.037 mg g−1 of seed dry wt. at 30 WAF which increased to 0.175 mg g−1 at 33 WAF and later decreased slowly to 0.013 mg g−1 at the end of maturity. The chlorophyll content of completely matured pongamia seeds was slightly lower than that reported in Brassica napus (Jain et al., 2008). The variation in the chlorophyll content during seed development has been reported in Brassica napus (Eastmond et al., 1996) and Glycine max (Laszlo, 1991). The ultrastructure study of developing pongamia seed showed increase in the plastid number at 33 and 36 WAF which coincided with the chlorophyll content of the developing

seed (Pavithra et al., 2013). It has been reported previously that photosynthetic activity was positively correlated with chlorophyll content during seed development (Eastmond et al., 1996). Since the early stages of pongamia seed development coincided with the prevalence of cool temperature (20 ◦ C) in the environment (November–December), low temperature could be the reason for slow degradation of chlorophyll as observed by Ward et al. (1992). Abscisic acid induced degradation of chlorophyll during seed desiccation could be the other reason for fast degradation (Nakajima et al., 2012). Electrical conductivity (EC) decreased from 996.3 to 354.6 dS m−1 (P < 0.01) during 30–42 WAF (Fig. 5). As the electrical conductivity is an indirect measure of membrane integrity during seed development, high EC at early stages of seed development (30, 33, 36 WAF) indicated the poor cell membrane integrity. At 39 and 42 WAF, there was a rapid decrease in EC indicating the improvement in cell membrane integrity and increase in reserve material deposition. The result of the present study is in concurrence with that reported in Brassica oleracea (Guruswamy and Thiagarajan, 1998) and Phaseolus vulgaris (Ghassemi-Golezani and Mazloomi-Oskooyi, 2008). The electrical conductivity of the developing seeds is also dependent on the lignin content of seed coat, genotype and moisture content (Panobianco and Vieira, 1996).

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Fig. 4. Mineral nutrient content of seeds at different stages of Pongamia pinnata fruit development. (g) Carbon content, (h) nitrogen content, (i) zinc, (j) copper, (k) iron, (l) manganese, (m) potassium and (n) phosphorus (SED, standard errors of difference; CD, critical difference).

3.5. Crude fibre and ash content The crude fibre content consisting of cellulose and lignin increased significantly during 30–42 WAF (1.27–6.10%) (Fig. 5). The histochemical staining of developing pongamia seed demonstrated an increase in lignin content during maturity which coincided with the crude fibre content of the developing seed (Pavithra et al., 2013). The ash content of seed which includes both the major and minor mineral elements remained almost the same (2.76–3.24%). The ash content of pongamia seed at maturity was higher than that reported by Sangwan et al. (2010). However, some reports suggested that ash

content did not vary much during seed maturation phase in Arachis hypogaea (Promchote et al., 2008) and Vicia sativa (Samarah and Ereifej, 2009). 3.6. Amino acid composition In pongamia, aspartate (Asp), glutamate (Glu), leucine (Leu), glycine (Gly), serine (Ser), lysine (Lys) are the major amino acids, while histidine (His), arginine (Arg), cysteine (Cys), valine (Val), methionine (Met), phenylalanine (Phe), isoleucine (Ileu), threonine (Thr), alanine (Ala) occurred in traces (Table 1). Among the amino

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Fig. 5. The (o) phytic acid, (p) chlorophyll content, (q) electrical conductivity, (r) crude fibre, (s) and ash content of seeds at different stages of Pongamia pinnata fruit development (SED, standard errors of difference; cd, critical difference).

acids, Asp, Glu, Ser, His and Cys were detected in seeds at all stages of fruit development. The content of these amino acids varied with the stage of development. Glu and Cys contents increased at 42 WAF, Asp increased up to 39 WAF and decreased at 42 WAF; however Ser decreased with increase in maturity. On the other hand, Gly and Thr were not detected at early stages, while Tyr was not detected at any of the five stages. Met, Ileu, Leu and Pro were not detected at 30 WAF but detected at later stages and Lys was detected at 42 WAF only. Although, not detected at early stages, certain amino acids increased gradually with the age of the seed (for example, Val, Leu, Pro) and certain others decreased gradually with seed age (for example, Gly, Met, Phe). The level of accumulation of seed storage proteins is influenced by differences in the rates of metabolism of nitrogen source and their inter-conversion into other amino acids. The Asp is required as substrate for asparagine biosynthesis in cytosol and as a precursor of the pathway leading to biosynthesis of essential amino acids like Lys, Met, Thr and Ilu. Glu remained prominent throughout the seed development. The Ser content was very high at the initial stages of development whereas Gly was not detected. The Ser content decreased by 36 WAF whereas Gly was detected at 36 WAF and it further decreased at maturity. Ser was reported to be synthesized from 3-phosphoglycerate or two

molecules of glycine (Ho et al., 1999). Variation in Ala, Leu and Val content during seed development could be due to availability of common precursor pyruvate. Lys and Thr are the essential amino acids present in limiting amounts in legume crops. Lys was detected at maturity whereas Thr was detected only at 36 WAF and thereafter it was not detected due to its occurrence in traces. Since Lys, Met and Thr are from the same aspartate biosynthetic pathway, the possibility of Lys and Thr feedback inhibition of enzyme could the limiting factor (Zhu and Galili, 2003). The Met content decrease during pongamia seed development could be due to elevated temperature while the Cys content increased with temperature. These results are in agreement with that of previous studies on soybean seed where decrease in Met content at high temperature coincided with increase in Cys content (Carrera et al., 2011). Sulfur amino acid biosynthetic pathway involving the synthesis of Cys from serine and further synthesis of Met from Cys (Tabe and Droux, 2001) could be the reason for the decrease in Ser content at seed maturity. The later stages of pongamia seed development at elevated temperature (35 ◦ C, January–February), could help in Pro accumulation (Forde and Lea, 2007). The amino acid composition of the pongamia seed at the time of maturity varied with the proximate values as reported by Prakash and Misra (1988). Such a variation in amino acid profile

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207

Table 1 Amino acid profile of seeds at various stages of fruit development in Pongamia pinnata. Amino acid (␮M)

30 WAF

33 WAF

36 WAF

39 WAF

42 WAF

Asp Glu Ser His Gly Thr Ala Arg Tyr Cys Val Met Phe Ileu Leu Lys Pro

438.59 ± 0.53 449.14 ± 0.60 773.11 ± 0.10 32.17 ± 0.15 NDa ND 0.43 ± 0.02 ND ND 0.44 ± 0.04 0.15 ± 0.002 ND ND ND ND ND ND

397.84 ± 1.17 1528.94 ± 0.87 626.92 ± 0.87 311.69 ± 0.41 ND ND ND ND ND 0.14 ± 0.003 ND 1.31 ± 0.06 ND 8.37 ± 0.06 13.45 ± 0.41 ND 12.84 ± 0.06

344.22 ± 0.88 935.75 ± 0.63 377.76 ± 0.97 54.54 ± 0.46 83.84 ± 0.77 42.17 ± 0.81 ND ND ND 1.42 ± 0.08 0.46 ± 0.02 1.51 ± 0.11 0.32 ± 0.02 13.75 ± 0.29 15.22 ± 0.86 ND 12.4 ± 0.45

403.05 ± 1.14 1471.05 ± 1.09 400.88 ± 1.25 49.12 ± 0.75 43.44 ± 0.37 ND ND ND ND 2.26 ± 0.16 0.62 ± 0.02 0.46 ± 0.04 ND 7.5 ± 0.33 51.01 ± 1.24 ND 65.23 ± 0.31

353.07 ± 1.33 2142.3 ± 0.37 51.3 ± 0.49 10.31 ± 0.17 6.51 ± 0.06 ND ND 4.42 ± 0.46 ND 3.43 ± 0.48 0.81 ± 0.01 0.52 ± 0.02 1.13 ± 0.16 14.0 ± 0.70 61.94 ± 0.58 32.05 ± 0.91 156.74 ± 0.46

Asp, aspartic acid, Glu, glutamic acid; Ser, serine; His, histidine; Gly, glycine; Thr, threonine; Ala, alanine; Arg, arginine; Tyr, tyrosine; Cys, cysteine; Val, valine; Met, methionine; Phe, phenylalanine; Ileu, isoleucine; Leu, leucine; Lys, lysine; Pro, proline, a Not detected.

could be attributed to the geographical locations, where the plant grows. 4. Conclusions Generally, all pongamia pods borne on the inflorescence are harvested in bulk resulting in pods harvested at different stages of maturity. The immature pods might contain reduced seed reserve material and oil content with low germination efficiency. The present study documents the variation in biochemical parameters like lipid, protein, sugar, starch and mineral nutrients at different seed developmental stages. Further, the ideal time for harvesting pongamia fruits was recommended at 42 WAF when fruits are completely mature with maximum reserve material, seed germination and vigour. The pongamia seeds showed developmental characteristics typical of other legume seeds. Information obtained on pongamia seed development could be used for profiling of enzymes involved in the biosynthetic pathway of oil. Further research is required in pongamia to define the role of pod wall development and its role in nutrient transfer to the developing seed. Acknowledgments The authors acknowledge the financial support and facilities received from the Department of Agriculture, Karnataka State Biofuel Development Board, Government of Karnataka. Authors also thank the University of Agricultural Sciences, GKVK, Bengaluru and the Department of studies in Applied Botany, School of Biosciences, Kuvempu University, Shimoga for extending research facilities. Thanks are due to Dr. M. Vasundhara, Department of Horticulture, UAS, GKVK, Bengaluru for providing HPLC facility. Sincere thanks to Mr. Rajesh Kumar for his help during HPLC analysis. References Alonso, A.P., Goffman, F.D., Ohlrogge, J.B., Shachar-Hill, Y., 2007. Carbon conversion efficiency and central metabolic fluxes in developing sunflower (Helianthus annuus L.) embryos. Plant J. 52, 296–308. Andriotis, V.M.E., Pike, M.J., Kular, B., Rawsthorne, S., Smith, A.M., 2010. Starch turnover in developing oilseed embryos. New Phytol. 187, 791–804. Anon., 1969. Wealth of India: Raw Materials. Publication and Information Directorate, Council of Scientific and Industrial Research, New Delhi. Anon., 1990. AOAC, Official Methods of Analysis, 15th ed. Association of Official Analytical Chemists, Washington, DC. Bernhardt, D.N., Wreath, A.R., 1955. Colorimetric determination of phosphorus by modified phosphomolybdate method. Anal. Chem. 27, 440–441.

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