EFFECT OF FOLIAR CHAPERONE TM APPLICATIONS UNDER ELEVATED TEMPERATURES ON THE PROTEIN CONCENTRATIONS AND PHYSIOLOGICAL RESPONSES OF COTTON

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1 AAES Reserch Series 521 EFFECT OF FOLIAR CHAPERONE TM APPLICATIONS UNDER ELEVATED TEMPERATURES ON THE PROTEIN CONCENTRATIONS AND PHYSIOLOGICAL RESPONSES OF COTTON R.S. Brown nd D.M. Oosterhuis 1 RESEARCH PROBLEM Vribility of endotoxin expression nd/or concentrtion within trnsgenic cotton (Bt) vrieties hs been nd continues to be concern of cotton growers, reserchers, nd breeders. While not consistent problem, it cn cuse mjor economic problems. On occsions, trnsgenic cotton crops require dditionl lteseson insecticide sprys due to the probble lck of endotoxin expressed in the cotton plnt. This vrition in endotoxin efficiency could be due to the inctivtion of the introduced gene leding to reduction in the mount of toxin produced. It is speculted tht this lck of endotoxin production my be due to incresed environmentl stresses such s elevted tempertures nd high light intensity. Informtion on the impct of environmentl stresses (prticulrly elevted tempertures) on protein nd endotoxin production is needed in order to mke mngement decisions surrounding current trnsgenic cotton lines nd for the development of new ones. From mngement perspective, pplictions of Chperone during elevted tempertures might be mens of improving protein nd endotoxin concentrtions nd thereby enhncing yields from incresed mortlity of bollworms. BACKGROUND INFORMATION Chperone is new Protein Enhncer for trnsgenic plnts, registered by the EPA in 2000, nd hs ptent pending. Chperone is combintion of nitrophenols, nmely sodium 5-ntroquicolte, sodium 0-nitrophenolte, nd sodium p-nitrophenolte. Phenolics ply centrl role in plnt metbolism nd growth nd they re known to increse photosynthetic electron trnsport, improve/ protect membrne integrity, nd increse enzyme/protein production (Robinson, 1980). Observtions in trnsgenic cottons hve shown tht endotoxin levels hve occsionlly filed to be fully expressed under vrious conditions, including environmentl fctors nd vrietl differences, thus occsionlly leding to less efficient insect control nd yield losses. Our hypothesis is tht utiliztion of the phenolic properties of Chperone in trnsgenic cotton would id in lleviting non-expression of Cry1Ac (BOLLGARD TM by Monsnto) or combintion of 1 Grdute ssistnt nd distinguished professor, Crop, Soil, nd Environmentl Sciences Deprtment, Fyetteville, respectively. 108

2 Summries of Arknss Cotton Reserch 2003 Cry1Ac with Cry2Ab, which re the genes currently utilized for expression of the endotoxin protein Bcillus thuringiensis. It hs been observed tht reduction in the mount of expressed endotoxin occurs s plnts mture, leding to loss of efficcy in the ltter stges of the growing seson thus incresing the probbility of surviving pests which my develop immunity to the endotoxin (Greenplte, 1999; Benbrook nd Hnsen, 1997). The objective of this growth-chmber study ws to determine the efficcy of single folir ppliction of Chperone under elevted tempertures on lef protein concentrtions nd physiologicl responses of the cotton plnt. RESEARCH DESCRIPTION A growth-chmber study ws initited fll 2003 t the University of Arknss, in Fyetteville, to determine the effect of single ppliction of Chperone t pinhed squre on lef totl soluble protein concentrtion nd physiologicl responses under elevted tempertures. The study ws rrnged in completely rndomized design with two tretments replicted four times. The tretments included Chperone t 5 oz/a rte pplied t pinhed squre nd n untreted control. The djuvnt penetrtor plus t 0.05% V/V ws used long with the Chperone ppliction. Cotton (Gossypium hirsutum L.) cultivr ST 4892BR ws plnted in 2L pots filled with sunshine mix nd wtered with hlfstrength Hoglnd s nutrient solution s needed to mintin moisture nd plnt nutrient levels. The growth chmber ws set for 12-hour photoperiod, with initil dy/night tempertures of 30/25 o C nd reltive humidities of 60 to 80%. At pinhed squre, Chperone ws pplied to four plnts, which long with four control plnts, were moved to second growth chmber progrmmed with the sme dy length nd humidity s the first growth chmber but progrmmed for n elevted dytime temperture of 33 o C. Three dys fter trnsferring the control nd Chperonetreted plnts to the second chmber progrmmed for elevted tempertures, mesurements were mde on plnts from the two growth chmbers (i.e with contrsting tempertures). Mesurements included totl soluble lef protein, photosynthesis, chlorophyll fluorescence, nd membrne integrity. Following this set of mesurements, two dditionl sets of plnts grown in the originl growth chmber t 30/25 o C were spryed three dys prt nd moved to the second growth chmber progrmmed for elevted tempertures of 36/25 o C nd 39/25 o C. Plnts were initilly plnted three dys prt to insure tht mesurements t the different temperture regimes would be performed on plnts of the sme ge. This would prevent ny dded doubt tht the results were due to stge-dependent differences in plnt development nd not to tretment effects. 109

3 AAES Reserch Series 521 RESULTS AND DISCUSSION Totl Soluble Protein Concentrtions Lte-seson insecticide pplictions re often wrrnted due to the probble lck of expression of Bt endotoxin, especilly under high tempertures. It ws hypothesized tht folir-pplictions of Chperone my be vible mens for incresing the efficcy of endotoxin nd protein concentrtions under elevted tempertures. Results from the growth chmber experiment in 2003 indicted tht single ppliction of Chperone under elevted tempertures numericlly incresed totl lef protein compred to the untreted control (Fig. 1). At 33 o C, there ws no difference between the control nd Chperone tretments for providing higher protein levels. However, under the higher tempertures (i.e. 36 nd 39 o C) Chperone-treted plnts hd numericlly higher lef protein concentrtions. Lef Photosynthesis Mesured lef photosynthesis indicted no differences between the control nd Chperone-treted plnts for improved lef photosynthetic efficiency cross the rnge of tested tempertures (Fig. 2). This result ws not surprising given tht lef photosynthesis often times will not be reduced until tempertures bove 40 o C predominte. Lef Fluorescence Lef chlorophyll fluorescence ws significntly decresed from pplictions of Chperone t 33 o C, 36 o C or 39 o C (Fig. 3). This indictes tht Chperone-treted plnts were exhibiting less stress t the elevted temperture regimes, in support of the hypothesis tht Chperone pplictions lessen the detrimentl effects of high tempertures on photosynthesis. Lef Membrne Integrity Lef membrne lekge, mesure of cell integrity, helps to explin the potentil efficiency of the cotton plnt for mintining optiml cell metbolism essentil for production of the developing fruit lod. Results indicted tht Chperone numericlly improved cell integrity (decresed lef-membrne lekge) cross ll tempertures tested (Fig. 4). The positive effect of chperone on membrne lekge ws significntly compred to the untreted control t 36 o C but not t 33 o C or 39 o C (Fig. 4). PRACTICAL APPLICATION Folir-pplied Chperone under elevted tempertures ppers to be 110

4 Summries of Arknss Cotton Reserch 2003 vible mens for incresing protein concentrtion nd, therefore, the efficiency of endotoxin expression. Incresed protein nd endotoxin expression from Chperone pplictions under environmentl stress conditions is the probble resons for the incresed bollworm mortlity nd incresed lint yields tht hve occurred in our field trils (Oosterhuis nd Brown, 2003). In ddition, improved efficiency from folir pplictions of Chperone my lso improve the overll physiologicl functioning of the cotton plnt. LITERATURE CITED Benbrook, M. nd M. Hnsen Return to the stone ge of pest mngement. Proc. Plnt Pesticide Resistnce Mngement, A Public Meeting, Mrch 21, 1997, Wshington D.C. Greenplte, J.T Quntifiction of Bcillus thuringiensis insect control protein CryIAc over time in BOLLGARD TM cotton fruit nd terminls. Journl of Economic Entomology. 92: Oosterhuis, D.M. nd R.S. Brown Incresed efficcy of Bt cotton with Chperone TM. Summries of Cotton Reserch in Ark. Agri. Exp. St., Res. Ser. 507: Robinson, T The Orgnic Constituents of Higher Plnts, 4 th edition. Cordus Press: N. Amherst, Mss. 111

5 AAES Reserch Series 521 Lef Protein (ug/ml) UTC Chperone UTC Chperone Tretment UTC Chperone Fig. 1. Effect of Folir Applictions of Chperone Under Elevted Tempertures on Totl Lef Protein Concentrtions. Brs with the sme letter re not significntly different t P <0.05. Lef Pn (umol [CO 2]m -2 s UTC Chperone 17.4 UTC Chperone UTC Chperone Tretment Fig. 2. Effect of Folir Applictions of Chperone Under ElevtedTempertures on Lef Photosynthesis. Brs with the sme letter re not significntly different t P<

6 Summries of Arknss Cotton Reserch 2003 Fluorescence (Fv/Fm) b UTC Chperone UTC Chperone UTC Chperone Tretment Fig. 3. Effect of Folir Applictions of Chperone Under Elevted Tempertures on Lef Fluorescence. Brs with the sme letter re not significnlty different t P<0.05. Lekge (ua/cm 2 ) b 0 UTC Chperone UTC Chperone UTC Chperone Tretment Fig. 4. Effect of Folir Applictions of Chperone Under Elevted Tempertures on Lef membrne Lekge. Brs with the sme letter re not significnlty different t P<