Lopes, Nei Fernandes

Marenco, Ricardo Antonio

Moreira, Maurílio Alves

Repositório do INPA

PHOTOSYNTHESIS AND LEAF LIPOXYGENASE ACTIVITYIN SOYBEAN GENOTYPES LACKING SEEDLIPOXYGENASE ISOZYMES1RICARDO ANTONIO MARENCO2, NEI FERNANDES LOPES3 and MAURILIOALVES MOREIRA3Departamento de Biologia Vegetal - Núcleo de Biotecnologia Aplicada àAgropecuária (BIOAGRO), Universidade Federal de Viçosa, Viçosa, MG,36570-000, Brazil.ABSTRACT- Plants from eight soybean genotypes, includinga triple null line (TNL) without seed lipoxygenase isozymes,were grown in a greenhouse to evaluate the effect of geneticelimination of seed lipoxygenases (LOX) on carbonassimilation and leaf LOX activity. Specific leaf matter,carbohydrate and chlorophyll concentration were assessed inplants from the TNL whose floral buds had been continuouslyremoved. There was no difference among genotypes on ratesof photosynthesis. Leaf LOX activity, however, was greater inthe genotype with the three seed LOX isozymes than in theTNL. The removal of floral buds in the TNL increased leafLOX activity, as well as, leaf carbohydrate and chlorophyllconcentration as compared to control plants, allowed todevelop seeds; the contrary was true for the rates ofphotosynthesis.Additional index terms: enzyme, carbohydrates, chlorophyll.FOTOSSÍNTESE E ATIVIDADE DELIPOXIGENASES NAS FOLHAS DEGENÓTIPOS DE SOJA SEMLIPOXIGENASES NAS SEMENTESRESUMO- Oito genótipos de soja, incluindo um genótipotriplo nulo, sem LOX nas sementes, foram cultivados em casade vegetação para avaliar o efeito da eliminação genéticadas LOX das sementes na fotossíntese e na atividade daslipoxigenases das folhas. A massa foliar específica e aconcentração de carboidratos e clorofila foram determinadosem plantas do genótipo triplo nulo (TNL) cujas flores tinhamsido removidas continuamente. Não houve diferenças entreos genótipos nas taxas de fotossíntese; a atividade das LOXdas folhas, entretanto, foi maior no genótipo com os três tiposde LOX nas sementes que no genótipo triplo nulo. As plantasdo genótipo TNL mantidas sem flores apresentaram maioratividade das LOX das folhas e maior concentração decarboidratos  e clorofila que aquelas  que frutificaramnormalmente, o contrário observou-se para as taxas defotossíntese.Termos adicionais para indexação: enzimas, carboidratos,clorofila.INTRODUCTIONLipoxygenases  (linoleate:oxygen oxidoreductase,EC 1.13.11.12) are dioxygenases that catalyze thehydroperoxidation of polyunsaturated fatty acidscontaining cis-cis pentadienes moieties. In higherplants, both linoleic and linolenic acids aresubstrate of LOX. In soybeans (Glycine max (L.)Merr.), these fatty acids are present in several planttissues, especially in the seeds (Siedow, 1991).Seeds of  soybeans contain at least three differentkinds of lipoxygenase isozymes, called LOX-1 (L-1),LOX-2 (L-2) and LOX-3 (L-3) (Siedow, 1991) whichappear to be different from those found in the leaves(Grayburn et al., 1991). Lipoxygenases in soybeanseeds promote the peroxidation of polyunsaturatedfatty acids which contributes to the  formation  ofundesirable flavor in soybean products.At the present, a precise physiological function ofLOX has not been entirely established, although theenzyme has been involved in growth anddevelopment, senescence, and also in plantresponses to infection with pathogens (Kato et al.,1992). Further, the study of LOX is receiving renewedinterest since they have been involved in jasmonicacid synthesis (Siedow, 1991). Jasmonic acidmarkedly increases the expression of specific genes,some of which are wound responsive (Staswick,1992). Depodding of soybean plants  may increase1 Recebido em09/08/94 e aceito em 15/04/1995.2 Eng. Agr. D.Sc., Prof. Visitante, Dpto. de Fitotecnia e Fitossani-dade, Univ. Estadual do MA, CP. 09, São Luís, MA, 65000-000,Brasil.3 Eng. Agr., Ph.D, Prof. Titular, Depto. de Biol. Vegetal, UFV,Viçosa, MG, 36570-000.3 Eng. Agr., Ph.D., Prof. Titular, BIOAGRO, UFV, Viçosa, MG,36570-000.21R.Bras.Fisiol.Veg., 7(1):21-25, 1995.the levels of leaf carbohydrate and  therefore,  therates of photosynthesis may be reduced (Mondal etal., 1978). Whereas removal of pods showed noeffects on specific leaf LOX activity in soybean(Tranbarger et al., 1991).Some genotypes lacking seed lipoxygenases havebeen obtained at the Universidade Federal deViçosa, becoming necessary to investigate theeffects of removing seed LOX on leaf LOX activity. Inthis  work,  leaf  LOX  activity  and photosynthesis insoybean genotypes lacking seed lipoxygenases wereinvestigated. Further, in a soybean seedlipoxygenase null line, photosynthesis, leaf LOXactivity, levels of leaf carbohydrates and chlorophyllcontent were studied in plants whose floral buds hadbeen removed.MATERIALS AND METHODSExperiment 1 - Eight soybean genotypes were in-cluded in this study: The commercial cv Cristalina withseed LOX, and seven genotypes from the Universi-dade de Federal de Viçosa germplasm collection, de-signed -L1, null for L-1; -L2, null for L-2; -L3, null forL-3; -L1L2, null for L-1 and L-2; -L1L3, null for L-1and L-3; -L2L3 null for L-2 and L-3, and a line lackingthe three seed LOX (-L1L2L3), hereinafter referred toas 'triple null line' (TNL). The plants were grown in agreenhouse under a 16 h photoperiod and about25/19oC day/night temperatures, in 4 L pots contain-ing fertilized potting compost. They were watered asnecessary with tap water to prevent water stress.Enzyme activity was measured in leaves collectedfrom plants at the V3 growth stage (Fehr et al., 1971).Middle leaflets of the second upper fully expandedtrifoliate  leaves  were collected at  the midmorning,weighed, immediately frozen in liquid nitrogen, andstored at -80oC until processing (Grimes et al., 1992).The tissue was ground to a powder in liquid nitrogenwith chilled mortar and pestle. Following grinding, thetotal soluble protein was extracted in bidistilled water,2 mL:1 g of tissue (Grayburn et al., 1991). Thehomogenate was centrifuged at 12,500 g for 15 minat 4oC  and  the  supernatant, referred  to  as  crudeextract, was assayed for LOX activity, at 23oC,based on the spectrophotometric methods of Axelrodet al. (1981). The standard assay mixture (1.03 mL)consisted of 1 mL of sodium phosphate buffer (50mM, pH 7.0); 5 µL of crude extract and 25 µL of asubstrate emulsion (10 mM linoleic acid emulsifiedin 0.36% Tween-20). The protein content in thesample was determined (Tombs et  al.,  1959), andthen the substrate was added to initiate the enzymereaction. The formation of hydroperoxides wascalculated by using 25,000 M-1cm-1 as the extinctioncoefficient. One unit of LOX activity, one katal, wasdefined as the amount of enzyme forming 1 mol ofhydroperoxide per min.Photosynthesis was measured at several plantgrowth stages. At V3 in the TNL and 'Cristalina'; atV12 in genotypes -L1, -L2, -L3, -L1L3, -L2L3, and atR5 in genotypes -L1L2 and in the TNL. It wasmeasured in the middle leaflet of the second upperfully expanded trifoliate leaf between 9:00 and 14:00under clear sky at a photon flux density of  1000µmol.m-2.s-1 or greater on a portableADC-LCA2/15001 infrared gas analyzer. Otherphotosynthesis  measurements were also taken atirradiance lower than 1000 µmol.m-2.s-1, between15:00 and 18:00, in the TNL and 'Cristalina' plants atthe V3 growth stage.Experiment 2 - Plants from the TNL were cultivatedas described above. At the beginning of flowering,they were randomly selected for floral bud removal(hereinafter referred to as the desinked plants), whilecontrol plants were allowed to flower and developpods and seeds. In desinked plants all the floral budswere removed twice weekly throughout plant devel-opment.The rates of photosynthesis were measured at theR5 growth stages between 9:00 and 14:00 h underclear sky, as described above. It was used the middleleaflet of the fifth trifoliate leaf beginning at the firsttrifoliate  leaf  from  the bottom  of  the  plant  in bothdesinked and control plants. In control plants, it wasverified that the leaf used for photosynthesismeasurements content at least one pod at its node.Further, photosynthesis, enzyme activity, specific leafmatter, chlorophyll and carbohydrates contents wereassessed at the same trifoliate leaf. Enzyme activityand  photosynthesis were measured in the middleleaflet, whereas specific leaf matter, chlorophyll andcarbohydrates were determined in lateral leaflets.The enzyme was assessed as described above,with some modifications, based on previous leaf LOXactivity assays (Marenco, R.A.; Lopes, N.F. &Moreira, A.A. - unpublished data). The standardmixture assay consisted of 1 mL of phosphate buffer(50mM, pH 6.6); 7.5 µL   of   substrate emulsion(10-mM linoleic acid emulsified in 0.36% Tween 20)and 2.0 µL of crude extract. Leaf LOX activity wasassayed at the V3, R4 and R5 growth stages in theTNL and only at the V3, V7 and V12 growth stagesin the cv Cristalina.Chlorophyll was determined by the method ofLichtenthaler  (1987) at the  V3, R5  and R6  growthstages in the  TNL  and at  the  V3 stage in  the cvCristalina. Specific leaf weight (SLW) was calculatedas SLW = LW/LA, where LW is the leaf dry matter,determined after drying at 70oC for 48 h and LA isthe leaf  area,  measured on a  Licor  (LI-3000) leaf22R.Bras.Fisiol.Veg., 7(1):21-25, 1995.area meter. Total soluble carbohydrates,  extractedfrom fresh leaflets in boiling 80% ethanol, werepurified with chloroform. The starch in the ethanolsoluble residue was extracted in 6.9% perchloricacid, being the precipitate removed by centrifugation(15 min, 5000 g). When necessary the samples werestored at -20oC until processing. Carbohydrateswere assessed spectrophotometrically at 620 nm,after their reaction with anthrone reagent (0.1% p/v,anthrone in sulfuric acid). Glucose was used as thestandard. The starch content in the sample wascalculated by multiplying the observed value by 0.9.RESULTS AND   DISCUSSIONExperiment 1 - Leaf LOX activity was lower in thegenotypes: -L1, -L2L3, and TNL than in 'Cristalina'(Fig. 1). The higher leaf LOX activity observed in 'Cris-talina' as compared to the TNL plants suggests aneffect of seed LOX genetic elimination on leaf LOXactivity, which may be important for soybean produc-tion since lipoxygenases have been involved in plantresponse to pathogen attack (Kato et al., 1992;Rickauer, et al., 1992) and wounding (Siedow, 1991).No effect on photosynthesis rates was observed as aconsequence of genetic removal of seed LOX (datanot  shown). Even though there was  effect  of seedgenetic elimination on leaf LOX activity, all the geno-types tested, including the TNL, grew and developedwell, which suggests that no essential physiologicalrole appear to be associated to seed LOX isozymes.Siedow (1991) suggested that a compensatory regu-lation take place in mutant lines to increase the ex-pression of one cotyledon LOX isozyme when anotheris missing. Therefore, if anyTABLE 1- Rates of photosynthesis (µmol.m-2.s-1 )in 'Cristalina' and triple null line (TNL) plants at theV3 and R5 growth stage.Genotype GrowthstageLeafmeasured TreatmentCO2assimilation1'Cristalina' V3 2nd - 10.39 aTNL V3 2nd - 9.99 aTNL R5 5th control 2.86 bTNL - 5th desinked 1.29 c1/ Means followed by the same letter are not significantly differ-ent at P = 0.01 by the Duncan test.leaf LOX isozyme is missing as a consequence ofseed LOX genetic elimination, a regulatory mecha-nism could be activated so that growth and develop-ment may occur normally, as observed in this experi-ment.Experiment 2 - There was no difference in chlorophylland carbohydrates concentration between 'Cristalina'and the TNL at the V3 growth stage. In addition, thesame rates of photosynthesis were observed at eitherhigh or low levels of irradiance (data not shown),which verified the results obtained in experiment 1.Desinked TNL plants showed lower rates ofphotosynthesis than control plants (Table 1), whichwas probably due to the high levels of carbohydrates,especially starch, accumulated in  desinked  plants(Table 2). Inhibition of photosynthesis by sink removalis consistent with  previous  results  (Mondal et  al.,1978; Wittenbach,  1983). The activity of LOX washigher in TNL desinked than in control plants at bothR4 and R5 growth stage (Table 3). A greater leaf LOXactivity in desinked plant may be probably a plantdefense mechanism since jasmonic acid, asecondary product of lipoxygenase action uponlinolenic acid (Siedow, 1991), may induce proteaseinhibitor in distant untreated leaves (Staswick, 1992).FIGURE 1- Leaf lipoxygenase (LOX) activity in soy-bean plants at the V3 growth stage. The genotypesincluded were: 'Cristalina'(CR), with seed LOX, andseven genotypes lacking seed LOX, designed, -L1,null for LOX-1; -L2, null for LOX-2; -L3, null for LOX-3; -L1L2, null for LOX-1 and LOX-2; -L1L3, null forLOX-1 and LOX-3; -L2L3 null for LOX-2 and LOX-3,and -L1L2L3 (TNL), null for LOX-1, LOX-2 and LOX-3. (n=4; bar, SE).23R.Bras.Fisiol.Veg., 7(1):21-25, 1995.TABLE 3- Leaf lipoxygenase activity (Katals.kg-1protein) in desinked and control triple null lineplants, without seed lipoxygenases, at both R4 andR5 growth stages.Treatment GrowthstageLeafmeasured LOX activityControl R4 5th 0.070 aDesinked - 5th 0.118 bControl R5 5th 0.087aDesinked - 5th 0.125 b1/ Means followed by the same letter are not significantly differ-ent at P = 0.01 by the Duncan test.The removal of floral buds increased the specificleaf weight and chlorophyll content, mainly at the R6growth stage (Table 2). Higher values of specific leafweight in desinked  plants did  verify a  continuedphotosynthate production following floral bud removalas suggested by Wittenbach (1983). From this studyit may be concluded that genetic elimination of seedLOX appears to have no effect on the rates ofphotosynthesis. On the other hand, even though TNLplants grew and developed seeds normally, theyshowed lower level of leaf LOX activity, which may beimportant from a  physiological point of view, sincelipoxygenases catalyze  the synthesis of jasmonicacid.ACKNOWLEDGEMENTSTo Profs. Gonçalves de Barros and Maria Goreti deAlmeida Oliveira  for helpful  suggestions  regardingchemical analysis.REFERENCESAXELROD, B.; CHEESBROUGH, T.M. & LAAKSO, S.Lipoxygenases from soybeans. Methods inEnzymology , 71:441-451, 1981.FEHR, W. R.; CAVINESS, C. E.; BURMOOD, D. T. &PENNINGTON, J. S.    Stage of developmentdescriptions for soybeans, Glycine max (L.) Merrill.Crop Science , 11:929-931, 1971.GRAYBURN, W. S.; SCHNEIDER, G. R.;HAMILTON-KEMP, T. R.; BOOKJANS, G.; ALI, K. &HILDEBRAND, D. F. Soybean leaves contain multiplelipoxygenases. Plant Physiology, 95:1214-1218,1991.GRIMES, H.D.; KOETJE,  D.S. &  FRANCESCHI, V.R.Expression, activity, and cellular accumulation ofmethyl jasmonate-responsive lipoxygenase insoybean seedlings. Plant Physiology , 100:433-443,1992.KATO, T.; MAEDA, Y.; HIRUKAWA, T.; NAMAI, T. &YOSHIOKA, N. Lipoxygenase activity increment ininfected tomato leaves and oxidation product oflinoleic acid by its in vitro enzyme reaction.Bioscience, Biotechnology and Biochemistry ,56:373-375, 1992.LICHTENTHALER, H. K. Chlorophylls and carotenoids:pigments of photosynthetic biomembranes. Methodsin Enzymology , 148:350-382, 1987.MONDAL, M. H.; BRUN, W. A. & BRENNER, M. L. Effectsof sink removal on photosynthesis and senescence inleaves of  soybean (Glycine max L.) plants. PlantPhysiology , 61:394-397, 1978.RICKAUER, M.; BOTTIN, A. & ESQUERRÉ-TUGAYÉ, M.T. Regulation of proteinase inhibitor production intobacco cells by fungal elicitors, hormonal factors andmethyl jamonate. Plant Physiology andBiochemistry , 30:579-584, 1992.TABLE 2- Total soluble carbohydrate (TSC, g.m-2), starch (g.m-2), specific leaf matter (SLW, g.m-2), andchlorophyll (Chl, mg.m-2) in leaf of control and desinked triple null line plants, without seed lipoxygenases, attwo growth stage (GS).Treatment GS TSC Starch SLW Chl-a Chl-b1control R5 1.36 b 9.46 b 33.0 b 88.0 bc 73.0 abdesinked - 1.88 a 18.19 a 53.0 a 111.0  b 81.0 abcontrol R6 1.61 a 3.23 c 29.0 b 64.0   c 54.0 bdesinked - 1.65 a 16.96 a 49.0 a 158.0   a 105.0 a1/ Means followed by the same letter are not significantly different at P = 0.05 by the Duncan test.24R.Bras.Fisiol.Veg., 7(1):21-25, 1995.SIEDOW, J.N. Plant lipoxygenase: structure and function.Annual Review of Plant Physiology and PlantMolecular Biology , 42:145-188, 1991.STASWICK, P.E. Jasmonate, genes, and fragrant signals.Plant Physiology , 99:804-807, 1992.TOMBS, M.P.; SOUTER, F. & MACLAGAN, N.F. Thespectrophotometric determination of protein at 210 m.Biochemistry Journal , 73:167-171, 1959.TRANBARGER, T. J.; FRANCESCHI, V. R.;HILDEBRAND, D. F. & GRIMES, H. D. The soybean94-kilodalton vegetative storage protein is alipoxygenase that is localized in paraveinal mesophyllcell vacuoles. Plant Cell , 3:973-987, 1991.WITTENBACH, V. A. Effect of pod removal on leafphotosynthesis and soluble protein composition offield-growth soybeans. Plant Physiology ,73:121-124, 1983.25R.Bras.Fisiol.Veg., 7(1):21-25, 1995.

Photosynthesis and leaf lipoxygenase activity in soybean genotypes lacking seed lipoxygenases isozymes

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Photosynthesis and leaf lipoxygenase activity in soybean genotypes lacking seed lipoxygenases isozymes

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