582 Roy et al.vp Acta Bot. Croat. 72 (1), 23–33, 2013 CODEN: ABCRA 25 ISSN 0365–0588 eISSN 1847-8476 Amino acids through developmental stages of sunflower leaves NAYAN ROY1, SUBRATA LASKAR2, ANANDAMAY BARIK1* 1 Ecology Research Laboratory, Department of Zoology, The University of Burdwan, Burdwan 713 104, West Bengal, India 2 Department of Chemistry, The University of Burdwan, Burdwan 713 104, West Bengal, India Abstract – The PICO-TAG analysis of proteins revealed that 17 protein-bound and 18 free amino acids were present throughout the developmental stages of sunflower leaves. The total protein-bound amino acid content was much higher than total free amino acid content throughout the development of sunflower leaves. The contents of protein-bound and free amino acids as well as essential and non-essential ones displayed different patterns with leaf maturation, suggesting that total protein levels are poor predictors of the nutritive status of leaves. Key words: amino acids, Helianthus annuus, sunflower Introduction Studies in plant-insect interactions have mainly concentrated on the effects of insect performance of plant secondary metabolites or nutritive compounds (SCHOONHOVEN et al. 2005). The nutritional quality of plants is generally characterized in terms of total nitrogen or protein content (RUUHOLA et al. 2003), and the growth efficiency of a variety of insects is closely related to plant nitrogen content. Further, the nutritive quality of plant tissues for insects may be affected by the amino acid composition of protein (SCHOONHOVEN et al. 2005). Low protein level in the insect diet, i.e., poor food resources from a nutritional point of view possibly may lead to ingestion of some amount of toxic secondary compounds (BROADWAY and DUFFEY 1988, HAUKIOJA et al. 1991, SLANSKY and WHEELER 1992, RUUHOLA et al. 2003), which may affect optimal insect growth, survival and fecundity. The balance of amino acids that constitute plant proteins differs from that of insects and deficiency in even one essential amino acid in a herbivore diet may cause an unbalanced nitrogen metabolism in insects (BERENBAUM 1995). ACTA BOT. CROAT. 72 (1), 2013 23 * Corresponding author, e-mail: anandamaybarik@yahoo.co.in Copyright® 2013 by Acta Botanica Croatica, the Faculty of Science, University of Zagreb. All rights reserved. 582 Roy et al.ps U:\ACTA BOTANICA\Acta-Botan 1-13\582 Roy et al.vp 14. o ujak 2013 10:34:15 Color profile: Generic CMYK printer profile Composite 150 lpi at 45 degrees Diacrisia casignetum Kollar (Lepidoptera: Arctiidae) is polyphytophagous and damages numerous field crops (i.e., sunflower, jute, sesame, castor, etc.) in India and many other Asian countries (ROY and BARIK 2012 a, b). It has been a serious pest of the sunflower (Heli- anthus annuus L.) in India for many years (BANERJEE and HAQUE 1984). It feeds gregari- ously on sunflower leaves leaving the mid ribs only. Variation in performance and abund- ance of phytophagous insects is mainly due to variation in qualitative and quantitative amounts of amino acids among host plants, including changes in the nutritional quality of leaves within a particular host plant during its different developmental stages (SCHOON- HOVEN et al. 2005). Herbivores often show a preference for young leaves within a plant because of the increasing toughness along with decreased water and protein content of mature leaves (MATTSON and SCRIBER 1987; SCHOONHOVEN et al. 2005). Further, the amino acid composition of the proteins indicates the nutritive quality of plant tissues, which may affect insect’s growth and development because total protein levels are poor predictors of the nutritive status of leaves (BERENBAUM 1995). Therefore, the present investigation was undertaken to determine the qualitative and quantitative variations in free and protein- -bound amino acids in sunflower (Helianthus annuus L.) leaves throughout the develop- mental stages of sunflower leaves, which will be used as experimental diets in further studies of the herbivorous insect, D. casignetum to understand the nutritional ecology of this insect pest for the purpose of developing better control strategies. There have been a number of studies on the amino acid content of sunflower leaves (CABELLO et al. 2006, DULERMO et al. 2009, AGUERA et al. 2010); but the available data mainly focuses on metabolic changes during natural ageing in sunflower leaves (CABELLO et al. 2006), amino acid changes in sun- flower cotyledon during a necrotrophic fungus (i.e., Botrytis cinera) interaction (DULERMO et al. 2009) and leaf development in sunflower plants grown with varying nitrate concen- trations (AGUERA et al. 2010). But, there have been no reports on the differences in free and protein-bound amino acids throughout the developmental stages of sunflower leaves. Materials and methods Plant material Fresh young (1–2 weeks old), mature (2–4 weeks old) and senescent (5–7 weeks old) sunflower cv. PAC-36 leaves were harvested randomly during January, 2011 from sun- flower plants (cv. PAC-36) growing in the field near Chinsurah Rice Research Center (22° 53' N, 88° 23' E), West Bengal, India (ROY and BARIK 2012a). Total amino acid content measurement The variability of total amino acid content of sunflower leaves throughout the develop- ment state of sunflower leaves was estimated taking 1g each of fresh young, mature and senescent leaves by the method of MOORE and STEIN (1948). Each determination was repeated three times. One gram of each kind of fresh leaf was placed separately in a hot-air oven at 50 ± 1 °C temperature for 72 h, materials were removed from the oven, and weighed in a digital monopan balance. One portion (25 mg) of the oven-dried sample was taken in a covered heat-resistant porcelain crucible (50 mL) and placed in a muffle furnace (Sunvic, UK) for burning. Material was initially allowed to smoke slowly and to lose organic matter gradually by increasing the furnace temperature at the rate of 5 °C min –1 to 450 °C and burnt 24 ACTA BOT. CROAT. 72 (1), 2013 ROY N., LASKAR S., BARIK A. 582 Roy et al.ps U:\ACTA BOTANICA\Acta-Botan 1-13\582 Roy et al.vp 14. o ujak 2013 10:34:15 Color profile: Generic CMYK printer profile Composite 150 lpi at 45 degrees to ash at 450 ± 5 °C for 30 min. The resultant ash was reconstituted with distilled water, dried to a constant dry weight in the hot air oven at 100 ± 0.5 °C for 2 h, and weighed in a digital monopan balance. This procedure was repeated three times for each kind of leaf tissue. The total amino acid content was presented as mean µg mg –1 ash-free leaf tissue ± standard error. Protein-bound amino acid measurement A sufficient amount of freshly collected leaves (young, mature and senescent) was rinsed with double distilled water and dried on a paper towel. One hundred g fresh leaf samples of each kind were dipped in 3 L n-hexane in a 5 L cotton-plugged conical flask and kept in the laboratory at room temperature for 21 days. The flask was then vigorously shaken daily for 30 min. The leaves were removed from n-hexane and dried in air at room temperature (27 ± 1 °C). The dried leaf material was extracted with phosphate buffer (pH 7) for 30 min, kept for 30 min in a –20 °C freezer, and was filtered through Whatman No. 41 filter paper (Maidstone, UK). Each kind of water extract was dialyzed in deionized water and then placed in a lyophilizer. The powdered protein obtained from each kind of leaf was weighed in a digital balance (± 0.01 mg). This process was repeated three times for each kind of leaf and values were expressed as mean ± standard error. These nine powdered protein samples were used for amino acid analysis separately. The powdered protein sample (20 µg) was hydrolyzed by 6N hydrochloric acid contain- ing 5% thioglycolic acid (MATSUBARA and SASAKI 1969). The solution was sealed in a tube under nitrogen and incubated in a hot-air oven at 110 °C for 24 h in the PICO.TAG work station. The hydrolyzed sample and the authentic amino acids internal standard – 'Standard H' (0.005 mL), were taken in respective tubes, introduced into the reaction vial and dried completely. These were then separately derivatised in a solution mixture of ethanol: triethyl amine: water: phenyl isothiocyanate (7: 1: 1: 1 v/v) in a nitrogen atmosphere at 25 °C for 20 min (GHOSH et al. 1997). The samples were dried and reconstituted in a diluent solution (Na2HPO4, 0.071% w/v in distilled water with pH 7.4; pH was adjusted by 10% H3PO4 containing 5% v/v acetonitrile). Amino acids were analyzed at 38 °C as per the PICO.TAG manual using a Pico-Tag C18 hydrophobic column (5µm, 3.9 × 150 mm; Waters) and detected at 254 nm (chart speed – 2 cm/min). Amino acids present in the unknown sample was characterized by comparing the peaks of the amino acids in the 'Standard H' (Pierce, Rockford, IL, USA), and the actual amount of each amino acid present was determined from the area under the individual curve. All solvents used were of analytical grade and purchased from E. Merck (India). Free amino acid measurement Freshly collected leaves of each kind (young, mature and senescent) were rinsed with double distilled water and dried by paper towel. One hundred g of fresh leaf samples of each kind were dipped in 2 L millipore water in a 5 L cotton-plugged conical flask and kept at room temperature (27 ± 1 °C) for 20 min using a magnetic stirrer. The extract of each kind was filtered through Whatman No. 41 filter paper. The filtrate was kept for 30 min in a –20 °C freezer and was again filtered through Whatman No. 41 filter paper (Maidstone, UK). The water extract from each kind of leaf was placed in lyophilizer. The powdered proteins obtained were weighed in a digital monopan balance. This procedure was repeated ACTA BOT. CROAT. 72 (1), 2013 25 AMINO ACIDS IN SUNFLOWER LEAVES 582 Roy et al.ps U:\ACTA BOTANICA\Acta-Botan 1-13\582 Roy et al.vp 14. o ujak 2013 10:34:16 Color profile: Generic CMYK printer profile Composite 150 lpi at 45 degrees three times for each of the samples of young, mature and senescent sunflower leaf. The total free amino acid content was presented as mean ± standard error. These powdered protein samples were used for amino acid analysis separately by the above mentioned procedure. Results Total amino acid content Total amino acid content varied throughout the developmental stages of sunflower leaves. Total amino acid content was the highest in mature leaves (3.61 ± 0.442 µg per mg ash-free leaf tissue) followed by young leaves (2.88 ± 0.378 µg per mg ash-free leaf tissue) and senescent leaves (2.287 ± 0.268 µg per mg ash-free leaf tissue). Amino acids bound in proteins Amino acid bound in proteins was greatest in mature leaves (0.521 ± 0.014 mg g–1 leaf tissue) followed by young leaves (0.441 ± 0.012 mg per g leaf tissue) and senescent leaves (0.383 ± 0.015 mg per g leaf tissue) (Tab. 1). The PICO.TAG analysis of protein-bound amino acids demonstrated that 17 different types of amino acids were present throughout the developmental stages of sunflower leaves (Tab. 2). The quantitative analysis revealed that bound monocarboxylic amino acids, aromatic amino acids, heterocyclic amino acids and sulphur-containing amino acids were present in highest level in young leaves, whereas dicarboxylic amino acids and hydroxy amino acids were present in the largest amount in mature and senescent sunflower leaves. The total monocarboxylic amino acid content grad- ually decreased throughout the developmental stages of sunflower leaves (from young leaf to senescent leaf). In this group, glycine was found to be absent during the development of sunflower leaves (Tab. 2). Further, alanine and leucine were present in the highest amount in young and mature leaves, respectively, whereas isoleucine was detected in trace amounts in senescent leaves. The two amino acids of the dicarboxylic group, aspartic acid and glutamic acid and their amides, were present in moderate amounts since they represent almost 20% of the total amino acids. Further, these two amino acids increased slightly throughout the developmen- tal age of sunflower leaves. The amount of hydroxy amino acids increased from young leaf (22.03 ± 0.814%) to mature leaf (34.02 ± 0.756%) and then slightly decreased in senescent 26 ACTA BOT. CROAT. 72 (1), 2013 ROY N., LASKAR S., BARIK A. Tab. 1. Total protein-bound and free amino acid content (mg per g fresh leaf tissue) throughout the developmental stages of sunflower leaves Leaf stages Protein-bound amino acids Free amino acids Young 0.441 ± 0.012 0.280 ± 0.007 Mature 0.521 ± 0.014 0.304 ± 0.010 Senescent 0.383 ± 0.015 0.226 ± 0.006 Mean ± SE, n = 3. 582 Roy et al.ps U:\ACTA BOTANICA\Acta-Botan 1-13\582 Roy et al.vp 14. o ujak 2013 10:34:16 Color profile: Generic CMYK printer profile Composite 150 lpi at 45 degrees ACTA BOT. CROAT. 72 (1), 2013 27 AMINO ACIDS IN SUNFLOWER LEAVES Tab. 2. Percentage of amino acids (g per 16 g N**) bound in proteins throughout the developmental stages of sunflower leaves Group Young Mature Senescent Monocarboxylic Alanine 11.14 ± 0.398 5.21 ± 0.133 3.44 ± 0.144 amino acids Glycine – – – Valine* 7.03 ± 0.300 6.59 ± 0.225 8.55 ± 0.292 Leucine* 9.60 ± 0.518 11.11 ± 0.543 8.18 ± 0.265 Isoleucine* 3.07 ± 0.132 4.14 ± 0.133 0.16 ± 0.012 Total 30.84 ± 0.277 27.05 ± 0.318 20.33 ± 0.159 Dicarboxylic Glutamic acid 10.40 ± 0.219 10.69 ± 0.318 11.78 ± 0.514 amino acids + glutamine Aspartic acid 9.40 ± 0.398 9.65 ± 0.179 11.14 ± 0.416 + asparagine Total 19.80 ± 0.618 20.34 ± 0.139 22.92 ± 0.098 Hydroxy amino Threonine* 7.13 ± 0.217 17.55 ± 0.497 17.48 ± 0.364 acids Serine 14.90 ± 0.537 16.47 ± 0.259 15.60 ± 0.248 Total 22.03 ± 0.814 34.02 ± 0.756 33.08 ± 0.612 Diamino acids Arginine* 3.37 ± 0.139 5.67 ± 0.352 3.66 ± 0.214 Lysine* 2.77 ± 0.058 3.56 ± 0.225 3.82 ± 0.156 Total 6.14 ± 0.081 9.23 ± 0.577 7.48 ± 0.369 Aromatic amino Tyrosine 5.25 ± 0.237 5.02 ± 0.398 5.65 ± 0.179 acids Phenylalanine* 8.66 ± 0.144 0.35 ± 0.017 5.92 ± 0.352 Total 13.91 ± 0.093 5.37 ± 0.381 11.57 ± 0.173 Heterocyclic Histidine* – – – amino acids Proline 1.29 ± 0.092 – 0.7 ± 0.011 Total 1.29 ± 0.092 – 0.7 ± 0.011 Sulphur containing Cysteine 0.79 ± 0.015 0.35 ± 0.023 0.43 ± 0.012 amino acids Methionine* 5.20 ± 0.012 3.64 ± 0.358 3.49 ± 0.163 Total 5.99 ± 0.005 3.99 ± 0.381 3.92 ± 0.176 Essential 46.83 ± 0.162 52.61 ± 0.109 51.26 ± 0.207 Non-essential 53.17 ± 0.162 47.39 ± 0.109 48.74 ± 0.207 * essential amino acid; Mean ± SE, n= 3. ** g per 16 g N = Amino acid composition data were reported as grams amino acid per 100 g of sample for each amino acid. The nitrogen content of the sample was used to convert amino acid per 16 g nitrogen and the values are expressed as g per 16 g N. For calculation of protein content, the nitrogen content is multiplied by 6.25, the practice originated from early research of proteins that were found to contain 16% nitrogen (100/16= 6.25). 582 Roy et al.ps U:\ACTA BOTANICA\Acta-Botan 1-13\582 Roy et al.vp 14. o ujak 2013 10:34:16 Color profile: Generic CMYK printer profile Composite 150 lpi at 45 degrees leaf (33.08 ± 0.612 %). Threonine was found to be present in the highest amount in mature (17.55 ± 0.497%) and senescent (17.48 ± 0.364%) leaves, whereas serine was present in the highest amount in young leaves (14.90 ± 0.537%) among all the amino acids. The total con- tent of diamino acids increased from young leaf to mature leaf and then decreased in senes- cent leaf, but this pattern is not followed in lysine content which increased slightly through- out the developmental stages of leaves. Aromatic amino acids were lower in mature leaves than young and senescent leaves. Ty- rosine was almost same throughout the developmental age of sunflower leaves, whereas phenylalanine drastically reduced to a trace amount from young leaf to mature leaf and then it increased almost seventeen fold in senescent leaf. In the heterocyclic amino acids group, histidine was found to be absent throughout the developmental state of leaves, whereas proline was absent in mature leaf. Among the sulphur-containing amino acids, cysteine was present in a small amount at all stages of leaf development, whereas methionine decreased from young leaf to senescent leaf. Free amino acids Total free amino acid content was highest in mature leaves (0.304 ± 0.010 mg g–1) fol- lowed by young leaves (0.280 ± 0.009 mg g–1) and senescent leaves (0.226 ± 0.006 mg g–1) (Tab. 1). The amount of the total monocarboxylic amino acids group gradually decreased throughout the development of sunflower leaves, like bound amino acids (Tab. 3). Though, the amount of this group was higher throughout the development of sunflower leaves in comparison with bound amino acids. Unlike bound amino acids, alanine was found to be absent, and glycine was present throughout the development stages of sunflower leaves. Further, glycine also formed a large portion of amino acids in mature leaves. The amount of aspartic acid and glutamic acid (and their amides) increased from young leaf to mature leaf stage and then a decrease was observed in senescent leaf (Tab. 3). The amount of hydroxy amino acid was lower throughout the developmental stages of sun- flower leaves in comparison with bound amino acids, but serine content was high in mature and senescent leaves in comparison with bound forms. The diamino acid content was higher than the bound amino acid content in young and senescent leaves. In mature leaves a de- creased free amino acid content was observed due to a drastic reduction of arginine. Tyrosine gradually decreased throughout the developmental stages of sunflower leaves. The percentage of phenylalanine was almost doubled from young leaf to senescent leaf ex- cept in the mature leaf where a large decrease (i.e., 2.26 fold from young leaf) was noticed. Phenylalanine was very high in mature and senescent leaves in comparison with the bound amino acid form. Histidine, which formed a major portion of heterocyclic amino acids in young and senescent leaves, was absent in bound amino acids. The percentage of proline content was almost equal in young and senescent leaf, but increased or decreased almost 3.5 fold in mature leaf from that in young leaf or senescent leaf, respectively. Among the sul- phur-containing amino acids, methionine followed almost the same pattern as the bound form whereas cysteine content was higher in the free amino acid form. The drastic decrease in the contents of essential free amino acids in mature leaves was due to an increase in the contents of non-essential amino acids. 28 ACTA BOT. CROAT. 72 (1), 2013 ROY N., LASKAR S., BARIK A. 582 Roy et al.ps U:\ACTA BOTANICA\Acta-Botan 1-13\582 Roy et al.vp 14. o ujak 2013 10:34:16 Color profile: Generic CMYK printer profile Composite 150 lpi at 45 degrees ACTA BOT. CROAT. 72 (1), 2013 29 AMINO ACIDS IN SUNFLOWER LEAVES Tab. 3. Percentage of free amino acids (g per 16 g N**) throughout the developmental stages of sunflower leaves Group Young Mature Senescent Monocarboxylic Alanine – – – amino acids Glycine 7.74 ± 0.243 20.69 ± 0.508 6.25 ± 0.248 Valine* 12.14 ± 0.490 6.51 ± 0.323 11.90 ± 0.473 Leucine* 9.48 ± 0.363 2.92 ± 0.207 4.97 ± 0.185 Isoleucine* 3.55 ± 0.133 2.26 ± 0.121 3.47 ± 0.121 Total 32.91 ± 0.249 32.38 ± 0.502 26.59 ± 0.658 Dicarboxylic Glutamic acid 8.82 ± 0.341 9.22 ± 0.266 4.22 ± 0.162 amino acids + glutamine Aspartic acid 2.43 ± 0.109 8.34 ± 0.213 3.84 ± 0.109 + asparagine Total 11.25 ± 0.450 17.56 ± 0.479 8.06 ± 0.271 Hydroxy amino Threonine* 1.19 ± 0.075 4.33 ± 0.167 1.27 ± 0.121 acids Serine 6.29 ± 0.265 23.28 ± 0.554 19.69 ± 0.487 Total 7.48 ± 0.341 27.61 ± 0.722 20.96 ± 0.609 Diamino acids Arginine* 12.17 ± 0.421 0.94 ± 0.012 10.77 ± 0.179 Lysine* 1.82 ± 0.185 1.06 ± 0.139 1.00 ± 0.040 Total 13.99 ± 0.236 2.00 ± 0.150 11.77 ± 0.219 Aromatic amino Tyrosine 9.48 ± 0.440 6.56 ± 0.150 3.19 ± 0.092 acids Phenylalanine* 6.66 ± 0.162 2.94 ± 0.080 13.83 ± 0.306 Total 16.14 ± 0.375 9.5 ± 0.231 17.02 ± 0.398 Heterocyclic Histidine* 10.85 ± 0.312 0.84 ± 0.017 9.61 ± 0.145 amino acids Proline 1.14 ± 0.069 4.17 ± 0.103 1.23 ± 0.069 Total 11.99 ± 0.381 5.01 ± 0.121 10.84 ± 0.214 Sulphur containing Cysteine 1.66 ± 0.133 2.03 ± 0.138 1.41 ± 0.133 amino acids Methionine* 4.58 ± 0.248 3.91 ± 0.133 3.35 ± 0.132 Total 6.24 ± 0.381 5.94 ± 0.272 4.76 ± 0.266 Essential 62.44 ± 0.092 25.71 ± 0.519 60.17 ± 0.216 Non-essential 37.56 ± 0.092 74.29 ± 0.519 39.83 ± 0.216 * essential amino acid; Mean ± SE, n= 3. ** g per 16 g N = Amino acid composition data were reported as grams amino acid per 100 g of sample for each amino acid. The nitrogen content of the sample was used to convert amino acid per 16 g nitrogen and the values are expressed as g per 16 g N. For calculation of protein content, the nitrogen content is multiplied by 6.25, the practice originated from early research of proteins that were found to contain 16% nitrogen (100/16= 6.25). 582 Roy et al.ps U:\ACTA BOTANICA\Acta-Botan 1-13\582 Roy et al.vp 14. o ujak 2013 10:34:16 Color profile: Generic CMYK printer profile Composite 150 lpi at 45 degrees Discussion The overall total amino acid concentrations in plants vary extremely, depending, how- ever, on environmental conditions (SHOBANA et al. 2010). Amino acid is an important factor affecting the feeding behaviour of herbivorous insects. The total content of protein-bound amino acids was much higher than that of total free amino acids throughout the develop- mental stages of sunflower leaves, indicating that the role of protein-bound amino acids is probably more important than that of free amino acids (RUUHOLA et al. 2003). The critical importance of amino acid composition of diet to the growth and reproduction of insects is well documented in the literature (HORIE and WATANABLE 1983, NATION 2001). While con- sidering variation in amino acid composition, it is important to distinguish among the deve- lopmental stages of plant leaves (KARLEY et al. 2002). This study demonstrated that changes in the contents of both free and protein-bound amino acids varied considerably throughout the development of sunflower leaves. The protein-bound amino acid content, i.e., dicarbo- xylic amino acids and hydroxy amino acids, is higher than free amino acid content through- out the developmental stages of sunflower leaves, whereas monocarboxylic amino acids, aro- matic amino acids, heterocyclic amino acids and sulphur containing amino acids are higher in free amino acids, indicating that the roles of free and protein-bound amino acids are both important in the nutrition of herbivores. A decrease in the contents of monocarboxylic pro- tein-bound or free amino acids occurred in senescing leaves which indicate the breakdown of cellular proteins and withdrawal of amino acids (RUUHOLA et al. 2003). Glycine was de- tected in large quantities in free amino acid forms in mature leaves, being the most abundant amino acid in this group. This amino acid, which is involved in many metabolic processes in the cell, apart from being a component in many proteins, was absent in bound forms. The relative levels of the two nitrogen-rich essential protein-bound amino acids, diamino acids, lysine and arginine, increased from young leaf to mature leaf and then decreased in senes- cent leaf. This decrease indicates the reduction of nutritive quality in senescent leaves (WEIBULL et al. 1990), since these two amino acids are target sites for proteolysis by trypsin which is a common protease of insect gut (BROADWAY and DUFFEY 1988). The high level of free amino acids in young and mature leaves, especially of free glutamic acid (and its amides), reflect the active metabolism of growing tissues (WEIBULL 1987). Interestingly, the relative content of free essential amino acids decreased at the expense of non-essential amino acids from young leaf to mature leaf in sunflower plants, suggesting that the quality of the amino acid pool actually decreased. Though the relative content of free essential amino acids increased again in senescent leaves, which is due to the higher content of histidine, arginine, valine and phenylalanine. Serine in free amino acid forms was found to be most active amino acid to promote senescence of leaves, while cysteine and phenyl- alanine had similar but less effect (MARTIN and THIMANN 1972). Senescent sunflower leaves also demonstrated a higher percentage of serine and phenylalanine than young leaves in free amino acid forms. Free aromatic amino acids are used for the synthesis of phenolic com- pounds and lignin (STRACK 1997). Further, phenylalanine is suggested to be a limiting factor for both the biosynthesis of phenolics and plant growth (JONES and HARTLEY 1999). The ab- solute content of free phenylalanine decreased 2.26 fold from young leaf to mature leaf and then increased almost five fold in senescent leaf. This suggests that phenylalanine is most active in senescent leaves and herbivorous insects do not prefer this kind of leaf. 30 ACTA BOT. CROAT. 72 (1), 2013 ROY N., LASKAR S., BARIK A. 582 Roy et al.ps U:\ACTA BOTANICA\Acta-Botan 1-13\582 Roy et al.vp 14. o ujak 2013 10:34:16 Color profile: Generic CMYK printer profile Composite 150 lpi at 45 degrees The process leading to developmental changes in amino acid composition might be due to developmental regulation of transporter expression from phloem loading of amino acids in leaf vascular tissue (FISCHER et al. 1995, KARLEY et al. 2002). However, there are other processes, i.e., metabolism, unloading and xylem-phloem transfer pathway which might be responsible for developmental changes of amino acid composition during leaf ageing (RENTSCH and FROMMER 1996, HIRNER et al. 1998, RUUHOLA et al. 2003). In the literature, clear information is available that the amino acid composition changes throughout the development of plant leaves (KARLEY et al. 2002, AMIARD et al. 2004). The reasons for this variation are probably related to fundamental aspects of plant physiology, i.e., the changes reflect the role of amino acid in both the form of nitrogen transported and the portioning of nitrogen during development (KARLEY et al. 2002). The value of leaves for its insect pests is known to decline rapidly with leaf maturation due to decrease in water and protein content as well as increased toughness of leaves (HAUKIOJA et al. 2002). But changes in the profiles of the protein-bound and free amino acids may further change the nutritive value of leaves because the dietary value of proteins may be inferior due to the absence of appropriate levels of needed amino acids (BRODBECK and STRONG 1987, SCHOONHOVEN et al. 2005). In conclusion, the amino acid composition of sunflower leaves in free and bound forms displayed different patterns throughout the developmental stages of sunflower leaves, which may provide useful information to clarify the quality of sunflower leaves as total protein levels are poor predictors for the nutrition for D. casignetum (RUUHOLA et al. 2003, SCHOONHOVEN et al. 2005). It will be interesting to follow the behavior of the herbivorous insect D. casignetum when feeding on the three stages of leaves with different quantities of amino acids in free and bound forms. Acknowledgement This study was supported financially by the University Grants Commission, New Delhi, India through a Minor Research Project (No. F. NO. 37/615/2009). References AGUERA, E., CABELLO, P., DE LA HABA, P., 2010: Induction of leaf senescence by low nitrogen nutrition in sunflower (Helianthus annuus) plants. Physiologia Plantarum 138, 256–267. AMIARD, V., MORVAN-BERTRAND, A., CLIQUET, J. B., BILLARD, J. 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