Mejo Int. J. Sci. Technol. 2011, 5(03), 365-373 Communiction Mejo Interntionl Journl of Science nd Technology ISSN 1905-7873 Aville online t www.mijst.mju.c.th Sunlight-stimulted phenyllnine mmoni-lyse (PAL) ctivity nd nthocynin ccumultion in exocrp of Mhjnk mngo Kokit Sengnil 1, 2, *, Knyrt Luengprsert 3 nd Jmnong Uthiutr 1, 2 1 Deprtment of Biology, Fculty of Science, Ching Mi University, Ching Mi 50200, Thilnd 2 Posthrvest Technology Innovtion Centre, Commission on Higher Eduction, Bngkok 10400, Thilnd 3 Biology Progrm, Fculty of Science nd Technology, Knchnuri Rjht University, Knchnuri 71000, Thilnd * Corresponding uthor, e-mil: kokit_s@hotmil.com Received: 7 Novemer 2010 / Accepted: 11 Novemer 2011 / Pulished: 14 Novemer 2011 Astrct: The ctivity of phenyllnine mmoni-lyse (PAL) required for nthocynin synthesis ws stimulted y sunlight exposure resulting in the development of red colour in Mhjnk mngo exocrp, which occurred only on the sunlight-exposed side of the fruit. The ccumultion of nthocynin ws concurrent with the increse in PAL ctivity in the mture stge of the fruit. The exposed side of the fruit hd higher PAL ctivity, endogenous sugr content, nd nthocynin ccumultion thn the. It is concluded tht sunlight increses red colour development of the mngo exocrp y inducing PAL ctivity. Exposure to sunlight lso enhnces endogenous sugr ccumultion in mngo fruit. Keywords: Mhjnk mngo, PAL ctivity, nthocynin in fruit exocrp, sunlight-induced nthocynin synthesis, endogenous sugrs INTRODUCTION Mhjnk mngo (Mngifer indic Linn. cv. Mhjnk) is hyrid cultivr etween Nng Klng Wn nd Sunset cultivrs in Ching Mi province, Thilnd. The fruit is elongted nd similr to Nng Klng Wn, with thick exocrp nd long edile portion of the thick yellowish-ornge mesocrp. It is soft nd hs good flvour nd thin pyrene, which mkes it suitle for consuming nd processing. The red colour of the exocrp is similr in intensity to
Mejo Int. J. Sci. Technol. 2011, 5(03), 365-373 366 Sunset mngo nd is due to nthocynins 1-3. However, Mhjnk mngo develops nonuniform red exocrp colourtion during fruit development. This reduces the vlue nd qulity of the fruit s it is uncceptle for foreign importers nd consumers in Thilnd. Light induces nthocynin synthesis nd red colour formtion in plnts. Sunlight increses the ctivity of the key enzymes for nthocynin synthesis, especilly phenyllnine mmoni-lyse (PAL), dihydroflvonol reductse (DFR), nthocynin synthse (ANS) nd UDP-glucose flvonoid 3-oglucosyltrnsferse (UFGT) 4-6. Light lso induces flvonoid synthesis involving severl photoreceptors such s phytochromes nd the photosynthetic system 6-7. In n experiment performed y Singh et l. 6, etiolted mize seedlings grown for six dys in drk conditions were exposed to sunlight for 4 hours nd trnsferred ck to drkness. Anthocynins were found to form in ll vegettive orgns with slow increse during 4-16 hours fter exposure to sunlight nd rpid increse fter 16-24 hours. PAL ws lso found to e stimulted with two distinct peks of ctivity: the first pek during 4-12 hours nd the second pek during 12-24 hours fter exposure. Sunlight intensity in different conditions during fruit development seems to ffect nthocynin ccumultion. As reported for pple (Mlus domestic) in Chin [8], the cultivrs Strkrimson (with red exocrp) nd Golden Delicious (with yellow exocrp) in highly irrdited re hd higher nthocynin ccumultion thn in the sme cultivrs grown in less irrdited re. In oth res, PAL ctivity in the red pple exocrp ws lso higher thn in the yellow one. Sunlight regultes nthocynin formtion in vegettive tissues nd orgns (root, mesocotyl, lef nd coleoptile). More nthocynin ccumultes in sun-exposed exocrp thn in shded one 7-9. In Elstr, Jongold, Elshof nd Red Elstr pples, the levels of cynidin 3-glctoside (nthocynin) nd quercetin 3-glycoside (flvonol) in the sun-exposed exocrp of individul fruit were much higher thn in the shded side 10. Anthocynin levels were highest in fruits orne on the top of the tree nd the outer tree prts, wheres the lowest levels of nthocynin were found in fruits from shded prts of the tree 7. Delicious pple fruits were covered with one-, two-, or threelyered pper gs to inhiit nthocynin ccumultion in the exocrp. After the gs were opened nd exposed to light for three dys, the fruit treted with the three-lyered g hd the highest nthocynin ccumultion 9. In Erly Blck crnerry fruits, nturl light conditions incresed totl nthocynin levels y 75.3 % nd 87.2 % fter 24 nd 48 hours respectively of wter immersion 11. Light indirectly increses nthocynin ccumultion y incresing endogenous sugrs, which induce the gene expression of key enzymes in nthocynin iosynthetic pthwys, especilly PAL enzyme. In Comet red rdish hypocotyls during cultivtion in light (~50 µe/m 2 s), totl sugrs (glucose, fructose nd sucrose) incresed in 21 dys fter sowing, nd the sugr ccumultion enhnced nthocynin production in the hypocotyl 12. In prticulr, sucrose-induced nthocynin production occurred vi incresed gene expression of chlcone synthse (CHS) nd nthocynidin synthse (ANS) in Comet red rdish hypocotyls with three times higher rtio of CHS:ANS expression from plnting until six dys fter 13. In the Incicle white rdish, the two nthocyninspecific genes (PAL nd CHS) responded to sucrose, nd other genes such s chlcone isomerse (CHI), flvnone 3-hydroxylse (F3H), DFR, nd ANS were wekly expressed, cusing less nthocynin ccumultion 13. When Lisinthus (Eustom grndiflorum cv. Royl Purple), potted
Mejo Int. J. Sci. Technol. 2011, 5(03), 365-373 367 plnt, ws cultured in high-intensity light (15,000 lux), the petls hd higher ccumultion of solule sugrs thn tht in low light intensity (1,000 lux), nd the high light intensity condition lso induced the expression of CHS, CHI nd DFR in nthocynin synthesis 14. In grpe erries (Vitis vinifer cv. Shirz) grown in sunlight, the totl solule solids of the fresh tissue incresed during 8-16 weeks fter flowering, long with n increse in nthocynin ccumultion in the erry exocrp fter 10 weeks. Expression of seven nthocynin iosynthesis genes (PAL, CHS, CHI, F3H, DFR, leuconthocynindin dioxygense (LDOX) nd UFGT) were enhnced during fruit development, especilly UFGT, which ws expressed only 10-16 weeks fter flowering nd coincided with nthocynin ccumultion in the erry exocrp 15. Sunlight lso promoted the synthesis of endogenous sugrs, which induced the gene expression for incresing nthocynin synthesis in V. vinifer cell suspension 16 nd Comet rdish hypocotyl 12. The two importnt fctors ffecting nthocynin synthesis nd ccumultion re light nd sugrs. Fructose nd sucrose were found to promote red colour development in the exocrp of Mhjnk mngo 2, while the involvement of sunlight in red colour development hs not een investigted. The ojective of this study is to investigte the effects of sunlight on PAL enzyme ctivity, sugr ccumultion, nd nthocynin ccumultion during the development of Mhjnk mngo exocrp. MATERIALS AND METHODS Plnt Stock Forty 7-yer-old Mhjnk mngo trees from n orchrd in Ching Mi province were used in this study. The experiment strted in Jnury nd continued until the end of My 2010. Inflorescences were tgged to record the ge of fruits from the dy fter full loom (DAFB). After tht, fruits t 70 DAFB (pproximtely 2 weeks prior to the eginning of red colour development in the fruit s exocrp s reported y Boonkn 1 ) which hung on the outer prt of the cnopy were smpled t 7-dy intervls until fruit mturity t 126 DAFB. The exocrp of ech fruit ws divided into 2 sides: the sunlight-fcing side nd the shded side; the former ws exposed to sunlight in the morning until middy. A totl of 360 fruits were used in this experiment. The freshly hrvested fruits were trnsported within 1 hour to Ching Mi University lortory for nlysis. The experiment followed completely rndomised design, with ech tretment eing pplied to 40 rndomly selected trees. Methods Extrction nd nlysis of PAL ctivity from the exocrp (1-mm thick) ws performed y method modified of those of Frgher nd Chlmer 17 nd Arkw et l. 18. The sornce ws compred with tht of stndrd cinnmic cid nd presented s nmole/mg protein hour. Protein quntity ws mesured ccording to the method of Lowry et l. 19 nd expressed s mg/g fresh weight. Extrction nd nlysis of reducing sugrs nd totl sugr content were determined y the modified Somogyi nd Nelson method 20-21 The sornce ws mesured t 540 nm nd
Mejo Int. J. Sci. Technol. 2011, 5(03), 365-373 368 compred with stndrd D-glucose. Determintion of totl nthocynin content ws conducted ccording to Rngnn 22. The sornce of the extrct ws mesured t 535 nm. The exocrp colours were mesured with colourimeter. The chromticity ws mesured s * vlue, which mesures the greenness nd redness on scle of -60 to +60. A minus * vlue mens green colour nd positive vlue, red colour. The sttisticl pckges for the socil science (SPSS) softwre for Windows ws used in this experiment for nlysis of vrince (ANOVA) nd lest-significnt difference (LSD) t 95% confidence level of ech vrile vlue under completely rndomised design (CRD). RESULTS PAL ctivity on the exposed side of mngo fruit exocrp incresed during two periods in fruit development t 91 nd 119 DAFB, nd ws significntly (p = 0.05) higher etween 84-126 DAFB thn the (Figure 1). Similr results were found on the, where PAL ctivity lso showed two peks, though not coinciding with those from the exposed side, t 77 nd 126 DAFB, nd ws lower thn tht on the exposed side (Figure 1). 30 PAL ctivity (nmole/mg protein.h) 25 20 15 10 exposed side Dys fter full loom Figure 1. Effect of sunlight on PAL ctivity of Mhjnk mngo fruit exocrp etween 70-126 DAFB. Brs with different letters re significntly different (p 0.05). Verticl rs indicte SE. The reducing sugr content of the exocrp incresed to the highest level t 91 DAFB nd then decresed slightly nd incresed gin t 126 DAFB (Figure 2). The exposed side hd higher reducing sugr content compred with the throughout fruit development (significnt t p = 0.05). The totl sugr content ws reltively constnt during 77-91 DAFB; fter tht it tended to increse. The highest content ws detected t 112 DAFB for the exposed side. Totl sugr content of the hd the sme trend throughout fruit development (Figure 3) ut ws found to e lower thn tht of the exposed side (significnt t p = 0.05).
Mejo Int. J. Sci. Technol. 2011, 5(03), 365-373 369 140 Reducing sugr content (mg/g dry weight) 130 120 110 100 90 exposed side 80 Dys fter full loom Figure 2. Effect of sunlight on reducing sugr content of Mhjnk mngo fruit exocrp etween 70-126 DAFB. Brs with different letters re significntly different (p 0.05). Verticl rs indicte SE. Totl sugr content (mg/g dry weight) 180 160 140 120 100 expose d side 80 Dys fter full loom Figure 3. Effect of sunlight on totl sugr content of Mhjnk mngo fruit exocrp etween 70-126 DAFB. Brs with different letters re significntly different (p 0.05). Verticl rs indicte SE. The totl nthocynin content of the exocrp significntly incresed on the exposed side s compred with the (Figure 4). Anthocynin content of the exposed side strted to increse t 84 DAFB nd rose to its highest level t 112 DAFB, which ws significntly higher thn tht of the. The totl nthocynin content of the remined reltively constnt etween 70-91 DAFB nd then grdully decresed.
Mejo Int. J. Sci. Technol. 2011, 5(03), 365-373 370 4 Totl nthocynin content (mg/100g fresh weight) 3 2 1 exposed side 0 Dys fter full loom Figure 4. Effect of sunlight on totl nthocynin content of Mhjnk mngo fruit exocrp etween 70-126 DAFB. Brs with different letters re significntly different (p 0.05). Verticl rs indicte SE. The chnge of * vlue of the exocrp ws investigted during fruit development (Figure 5). Red colourtion of the exposed side of the fruit ws first oserved t 84 DAFB nd the highest * vlue ws oserved t 112 DAFB, indicting mximum red colour development. The hd minus * vlue indicting green exocrp. The * vlues of ll tretments were significntly different throughout the fruit development period. 6 4 2 * vlue 0-2 -4-6 exposed side -8 Dys fter full loom Figure 5. Effect of sunlight on * vlues of Mhjnk mngo fruit exocrp etween 70-126 DAFB. Brs with different letters re significntly different (p 0.05). Verticl rs indicte SE.
Mejo Int. J. Sci. Technol. 2011, 5(03), 365-373 371 DISCUSSION An ccumultion of nthocynin in the sunlight-exposed side of Mhjnk mngo fruit exocrp ws concurrent with n increse in PAL ctivity in the mture stge t 105-126 DAFB ut not t 70-98 DAFB (Figures 1 nd 4). It is possile tht the first pek of PAL ctivity in the immture fruits is necessry for phenolic metolism tht is directed to the synthesis of simple phenols nd flvonoids during fruit development. However, the reltionship etween PAL ctivity nd nthocynin ccumultion vries ccording to the stge of fruit development, which is similr to wht occurs in pple exocrp 5, 8. Similr to our results, Awd et l. 7 found tht incresing nthocynin synthesis nd development of red colour in pple exocrp depend on sunlight. This is true in pples receiving sunlight from the top of the tree, which is exposed to higher light intensity. These fruits tend to hve more nthocynin synthesis thn those t other positions. Visile light (400-700 nm) nd lso UV-B (312 nm) hve the most importnt role in promoting red exocrp colour development in pples 23. A highly exposed plce increses the efficiency of nthocynin synthesis nd stimultes higher nthocynin ccumultion compred to shded re 8. Other studies hve shown tht the photosynthesis pthwy stimultes the synthesis of precursors for nthocynin production 23-24. In our experiment, sunlight lso stimultes n increse of reducing sugr nd totl sugr content lrgely on the exposed side of the exocrp, where the chnge in totl sugr content closely follows the chnge in nthocynin level except t 70-77 DAFB. Totl sugr content lso prllels PAL ctivity ut only in mture fruits. Trnsport of sugrs, especilly sucrose from the source (lef) to the sink (fruit), is stimulted y sunlight. Sucrose is trnslocted to fruit vi phloem cusing n increse in endogenous sugrs 25. This is consistent with the report of Gun nd Jnes 26, who found tht the mount of sugr in tomto fruit (Lycopersicon esculentum) ccumulted in light is higher thn in the drk. Light stimultes uptke, metolism nd storge of sugrs, especilly sucrose, which is mjor sugr trnslocted into the tomto fruit. Light-grown fruits hve higher crohydrte content thn drk-grown fruits, s well s higher ADP glucose pyrophosphorylse ctivity, which is correlted with high strch level in tomto fruit 27. Endogenous sugrs induce gene expression of PAL enzyme, the key enzyme in the nthocynin synthesis pthwy. According to Vitrc et l. 16, the result of Vitis vinifer cell suspension cultures showed tht sucrose promotes nthocynin production nd recognises the generl signl trnsduction such s C +, clmodulin nd protein kinse/phosphtses for inducing nthocynin iosynthesis. In red rdish hypocotyls treted with exogenous sucrose, the expression levels of PAL, CHS, CHI, F3H, DFR nd ANS for nthocynin iosynthesis were enhnced 13. In corn leves (cv. Suweon 19), sucrose, which is importnt for controlling CHS, CHI, F3H, DFR nd ANS, ws shown to produce the gretest stimultion of nthocynin formtion 28. CONCLUSIONS Sunlight induces red colour development in mngo fruit exocrp y promoting nthocynin synthesis vi incresing PAL ctivity. Sunlight lso increses endogenous sugrs (reducing sugrs nd totl sugrs) during mngo fruit development.
Mejo Int. J. Sci. Technol. 2011, 5(03), 365-373 372 ACKNOWLEDGEMENTS We would like to thnk Mr. J. F. Mxwell of the Biology Deprtment, Ching Mi University for reviewing nd improving the mnuscript. This work is supported y grnt from the Posthrvest Technology Innovtion Centre, Commission on Higher Eduction, Bngkok. The Deprtment of Biology, Ching Mi University is cknowledged for providing the fcilities, chemicls nd equipment needed in this work. REFERENCES 1. S. Boonkn, Physiologicl nd chemicl chnges during growth nd development of Mhjnk mngo fruit, MS Thesis, 2002, Ching Mi University, Thilnd. 2. K. Luengprsert, J. Uthiutr, K. Sengnil nd O. Arkw, The effects of sugr ppliction on the concentrtions of nthocynin nd flvonol of Mhjnk mngo (Mngifer indic Linn. cv. Mhjnk) fruit, Ching Mi J. Sci., 2010, 37, 355-362. 3. G. Mzz nd E. Miniti, Anthocynins in Fruits, Vegetles, nd Grins, CRC Press, Boc Rton, 1993. 4. J. E. Lncster nd D. K. Dougll, Regultion of skin color in pples, Crit. Rev. Plnt Sci., 1992, 10, 487-502. 5. Z. G. Ju, Y. B. Yun, C. L. Liou nd S. H. Xin, Reltionships mong phenyllnine mmonilyse ctivity, simple phenol concentrtions nd nthocynin ccumultion in pple, Sci. Hortic., 1995, 61, 215-226. 6. A. Singh, M. T. Selvi nd R. Shrm, Sunlight-induced nthocynin pigmenttion in mize vegettive tissues, J. Exp. Bot., 1999, 50, 1619-1625. 7. M. A. Awd, P. S. Wgenmkers nd A. de Jger, Effects of light on flvonoid nd chlorogenic cid levels in the skin of Jongold pples, Sci. Hortic., 2001, 88, 289-298. 8. X. J. Li, J. H. Hou, G. L. Zhng, R. S. Liu, Y. G. Yng, Y. X. Hu nd J. X. Lin, Comprison of nthocynin ccumultion nd morpho-ntomicl fetures in pple skin during color formtion t two hitts, Sci. Hortic., 2004, 99, 41-53. 9. Z. Ju, Fruit gging, useful method for studying nthocynin synthesis nd gene expression in pples, Sci. Hortic., 1998, 77, 155-164. 10. M. A. Awd, A. de Jger nd L. M. vn Westing, Flvonoid nd chlorogenic cid levels in pple fruit: chrcteristion of vrition, Sci. Hortic., 2000, 83, 249-263. 11. Y. Zhou nd B. R. Singh, Effect of light on nthocynin levels in sumerged, hrvested crnerry fruit, J. Biomed. Biotechnol., 2004, 5, 259-263. 12. M. Hr, K. Oki, K. Hoshino nd T. Kuoi, Enhncement of nthocynin iosynthesis y sugr in rdish (Rphnus stivus) hypocotyl, Plnt Sci., 2003, 164, 259-265. 13. M. Hr, K. Oki, K. Hoshino nd T. Kuoi, Effects of sucrose on nthocynin production in hypocotyl of two rdish (Rphnus stivus) vrieties, Plnt Biotechnol., 2004, 21, 401-405. 14. S. Kwt, Y. Kusuhr, Y. Li nd R. Skiym, The regultion of nthocynin iosynthesis in Eustom grndiflorum under low light conditions, J. Jpn. Soc. Hort. Sci., 1999, 68, 519-526.
Mejo Int. J. Sci. Technol. 2011, 5(03), 365-373 373 15. P. K. Boss, C. Devies nd S. P. Roinson, Anlysis of the expression of nthocynin pthwy genes in developing Vitis vinifer L. cv Shirz grpe erries nd the implictions for pthwy regultion, Plnt Physiol., 1996, 111, 1059-1066. 16. X. Vitrc, F. Lrronde, S. Kris, A. Decendit, G. Deffieux nd J. M. Merillon, Sugr sensing nd C +2 -clmodulin requirement in Vitis vinifer cells producing nthocynins, Phytochem., 2000, 53, 659-665. 17. J. D. Frgher nd D. J. Chlmers, Regultion of nthocynin synthesis in pple skin. III. Involvement of phenyllnine mmoni-lyse, Aust. J. Plnt Physiol., 1977, 4, 133-141. 18. O. Arkw, Y. Hori nd R. Ogt, Chrcteristics of colour development nd reltionship etween nthocynin synthesis nd phenyllnine mmoni-lyse ctivity in Strking Delicious, Fuji nd Mutsu pple fruits, J. Jpn. Soc. Hort. Sci., 1986, 54, 424-430. 19. O. H. Lowry, N. J. Roserough, A. L. Frr nd R. J. Rndll, Protein mesurement with the folin phenol regent, J. Biol. Chem., 1951, 193, 265-275. 20. M. Somogyi, Notes on sugr determintion, J. Biol. Chem., 1952, 195, 19-23. 21. N. J. Nelson, A photometric dpttion of the Somogyi method for the determintion of glucose, J. Biol. Chem., 1944, 153, 375-380. 22. S. Rngnn, Plnt pigments, in Mnul of Anlysis of Fruit nd Vegetle Products (Ed. S. Rngnn), Tt McGrw-Hill Pulishing Co., New Delhi, 1977, pp. 72-93. 23. O. Arkw, Y. Hori nd R. Ogt, Reltive effectiveness nd interction of ultrviolet-b, red nd lue light in nthocynin synthesis of pple fruit, Physiol. Plnt., 1985, 64, 323-327. 24. O. Arkw, Photoregultion of nthocynin synthesis in pple fruit under UV-B nd red light, Plnt Cell Physiol., 1988, 29, 1385-1389. 25. W. G. Hopkins nd N. P. A. Hüner, Introduction to Plnt Physiology 4 th Edn., John Wiley nd Sons, Dnvers, 2009. 26. H. P. Gun nd H. W. Jnes, Light regultion of sink metolism in tomto fruit. I. growth nd sugr ccumultion, Plnt Physiol., 1991, 96, 916-921. 27. H. P. Gun nd H. W. Jnes, Light regultion of sink metolism in tomto fruit. II. crohydrte metolizing enzymes, Plnt Physiol., 1991, 96, 922-927. 28. J. H. Kim, B. H. Lee, S. H. Kim, K. H. Oh nd K. Y. Cho, Responses to environmentl nd chemicl signls for nthocynin iosynthesis in non-chlorophyllous corn (Ze mys L.) lef, J. Plnt Biol., 2006, 49, 16-25. 2011 y Mejo University, Sn Si, Ching Mi, 50290 Thilnd. Reproduction is permitted for noncommercil purposes.