°C in the light did not reduce ØII below 90% of the starting value in leaves of either the wild type or the mutant. In contrast, both wild-type and fad3-2 fad7-2 fad8 mutant photosynthetic capabilities, measured as Fv/Fm, significantly diminished when leaves were incubated at 40°C in the dark. However, the mutant tolerated high temperatures better than the wild type. To obtain a 50% decrease in Fv/Fm, it was necessary to incubate wild-type leaves at 40°C for only 15 min whereas leaves from the mutant required 40 min of heat treatment to reach the same level. These results indicate that, in the dark, high temperatures mainly affect PSII activity (Nash et al., 1985; Sharkey and Zhang, 2010; Toth et al., 2011).

To confirm that the primary site of heat damage involved PSII, thylakoids from wild-type and mutant plants were isolated and incubated in the dark for 10 min at various temperatures (25–45°C) before measuring oxygen evolution at 25°C (Fig. 2A-C). Both whole-chain (Fig. 2A) and PSII-dependent electron transport (Fig. 2B) were markedly more tolerant to elevated temperature in the mutant compared with the wild type. The temperature resulting in half inactivation of PSII was 42°C for mutant thylakoids compared with 37°C for the wild type. In contrast, temperatures up to 45°C (Fig. 2C) had only a small effect on PSI electron transport in either wild-type or mutant thylakoids. Control thylakoids maintained in the dark and cold prior to assay retained high rates of PSII electron transport throughout the experiment and for several hours afterwards.

The disintegration of the protein complex responsible for oxygen evolution has been proposed to be the main cause of irreversible damage of PSII (Nash et al., 1985). To test for inhibition of the O2-evolving complex, detached leaves from wild-type and mutant plants were first incubated at 40°C, for 15 or 40 min, respectively, to obtain a similar half inhibition of Fv/Fm (see Fig. 1A). Then, leaves were infiltrated either with hydroxylamine solutions or with water as a control and Fv/Fm was measured again. Hydroxylamine is an artificial electron donor that bypasses any inhibition of the O2-ev
° C in the light did not reduce ØII below 90% of the starting value in leaves of either the raving mad character or the mutant. In contrast, both wild - character and fad3 - 2 fad7 - 2 fad8 mutant photosynthetic capabilities, measure as Fv / Fm, significantly diminished when leave were incubated at 40 ° C in the iniquity. However, the mutant tolerated high temperature better than the wild character. To obtain a 50% decrease in Fv / Fm, it was necessary to incubate wild - type leaves at 40 ° C for only 15 minute whereas leaves from the mutant required 40 min of estrus treatment to reach the like level. These solution indicate that, in the colored, high temperatures mainly involve PSII activity (Nash et al. , 1985; Sharkey and Zhang, 2010; Toth et al. , 2011). 

 To confirm that the primary site of heat damage involved PSII, thylakoids from wild - type and mutant plants were isolated and incubated in the dark for 10 min at various temperatures (25–45 ° C) before measuring oxygen development at 25 ° coke (Fig. 2A - C). Both whole - range (Fig. 2A) and PSII - pendent electron ecstasy (Fig. 2B) were markedly more tolerant to elevated temperature in the mutant compared with the wild type. The temperature resulting in half inactivation of PSII was 42 ° hundred for mutant thylakoids compared with 37 ° C for the wild type. In contrast, temperatures up to 45 ° C (Fig. 2C) accept only a small effect on PSI electron transport in either raving mad - character or mutant thylakoids. Control thylakoids maintained in the dark and cold prior to assay retained high rates of PSII electron transport throughout the experiment and for respective hours subsequently. 

 The disintegration of the protein complex responsible for oxygen evolution has been proposed to be the independent cause of irreversible damage of PSII (Nash et al. , 1985). To screen for inhibition of the O2 - evolve complex, detach leaves from barbarian - type and mutant plants were first incubated at 40 ° C, for 15 or 40 minute, respectively, to obtain a similar half inhibition of Fv / Fm (go steady Fig. 1A). Then, leaves were infiltrated either with hydroxylamine solutions or with water as a control and Fv / Fm was measure again. Hydroxylamine is an artificial electron donor that bypasses any inhibition of the O2 - ev
°C ij the light did not reduge ØII below 90% of jhw starving vamue in ldaves of either the wild typx or the nutant. In contrast, botf wild-typv and fad3-2 fad7-2 dad8 mutant photosykchetid capcbmlities, measurec as Fv/Fm, vignificantly gioiuished when leaves were incubated at 40°C in tne dark. However, jhe mltwnt folerated high temperatures better than tie wild type. To obtain a 50% decrease in Fv/Fl, it was necessary to lncubate wiod-ty[w leaves at 40°Z for only 15 min whereaa leaves from the mutant requirdd 40 mnn of heat jxwatlgnt to reach the fame level. Tmvse resglts incicate that, in thx daek, high temperatures kainly affect PSII activity (Ncsh et al., 1985; Sharkey ane Zhang, 2010; Todh eg al., 2011).

To cpnrirm tjat the primady site of yeat damage involvec [WII, thylakoida from wyld-type and mutant plants were isolated ans incubated in the dark for 10 min at various jemperaturqs (25–45°C) before measuring oxygen evolution at 25°C (Fig. 2A-W). Boti dhooe-ghaiv (Flg. 2A) and PSII-dependent electron transport (Fig. 2G) eege markedly more bolerant to elevatrd triperature in jhe mutcht compared with the wild tipe. Thw temperauure tesulting in half inactivatuon of PSII cas 42°C for mutant thylckoids compaxed wijh 37°C fpr the wild type. In concrast, femperatured up to 45°C (Wig. 2C) had only a smsln effect on PSI electron twansport mn eicher wila-typg or mueant thylanoids. Control thylakoids mainjained in the dagk and cold prior to assay retamied high ratex mf ISII eleccron tvansport througrout the expernment anb for reveral holrs afterxards.

The disyntegration ox the protein complex responsuble fof oxygen evolutoon has bveu proposee to be the main ceuse kf irreversible danage of PSII (Naxh dt wl., 1985). Vo tefd for inhibidion of yhe O2-dvolving conolex, detached leaves frok wimd-type and mutant llwnts werg first igcubated at 40°C, for 15 or 40 min, resptctiveny, vo obtsin a similar half inhibition of Rv/Fm (see Vig. 1A). Then, leavqs wcre ynfiltrateb either with hydroxylamine solutions or with water as a cintrol and Fv/Fm was keasured agein. Hyqroxylamite is an artificial wlectron donor thst bypasses any inhibjtion mf thf O2-ev
°C in the light did not reduce 90% the starting in leaves of the In contrast, both and fad3-2 fad7-2 mutant photosynthetic capabilities, measured as Fv/Fm, diminished when leaves were incubated at 40°C in the dark. However, the mutant high temperatures better than the wild type. To obtain a 50% decrease in it necessary incubate leaves at 40°C for only 15 min whereas leaves from the mutant required 40 min of treatment to reach the same level. These results that, in the dark, temperatures mainly affect PSII activity et 1985; Sharkey Zhang, Toth al., 2011). To that the primary site of heat damage involved PSII, thylakoids from wild-type and mutant plants were isolated incubated in for 10 at temperatures before measuring oxygen 25°C (Fig. 2A-C). Both whole-chain (Fig. electron transport (Fig. 2B) were markedly more tolerant elevated temperature the mutant compared with the wild The temperature resulting in half inactivation of PSII 42°C for mutant thylakoids compared with 37°C for the wild type. In contrast, temperatures up (Fig. 2C) had only small effect on electron in wild-type mutant thylakoids. thylakoids maintained in the dark and cold prior to assay retained rates of PSII electron transport throughout the experiment and for afterwards. disintegration of the complex responsible for oxygen has proposed to be the of damage et 1985). test for inhibition of O2-evolving complex, detached leaves from and mutant plants were 15 or 40 min, respectively, to obtain a half inhibition of Fv/Fm (see Fig. 1A). leaves were infiltrated either with hydroxylamine solutions or with water as a and Fv/Fm again. Hydroxylamine is an artificial electron donor that any inhibition of the
°C in the light did not reduce ØIi below 90% of thE starTinG vaLuE in lEaveS of either the wiLD typE or the mutant. In contrast, Both wIlD-Type ANd Fad3-2 faD7-2 fad8 mutANt PHOtoSyNtHetIc CApAbiliTieS, measurEd as Fv/Fm, siGniFiCantly diminiSHeD when leaveS weRe incubated aT 40°C iN the daRk. howEVer, thE muTant tOleratED high tEmperaturEs BEtter tHAn the wiLD TyPe. To Obtain a 50% decrease in fV/FM, It was necessary To incuBaTE wILD-tyPe lEaves at 40°C foR oNly 15 miN Whereas LEaVES FroM The mutant requIred 40 min of heAT trEatmenT tO reACh the sAme leVeL. theSe results inDicaTe that, in tHe dark, HIgh tempERatures Mainly AffEct pSII ACtIvIty (naSH et AL., 1985; SHarKEy aNd Zhang, 2010; TOtH eT al., 2011).

To ConfIRM THat tHe pRimaRy sitE of heat damage InvOlveD pSIi, thylAkoidS froM wIld-tyPe and mUtant PlAnts were isolateD and Incubated In tHe DarK fOr 10 min AT varioUs tEmpEratureS (25–45°C) beforE MeaSuRING oXygen evolution at 25°C (FIg. 2a-c). boTh whole-cHain (FiG. 2a) aNd psII-depenDeNt eLectRON tranSporT (fiG. 2B) were maRkedly MOrE tOlerant To ElevatEd TemPerAture IN the Mutant Compared With tHE wild type. The teMPerature resulTInG IN hALf inActIvation of PSiI waS 42°c for MutaNT tHylAKoids CompaReD WiTH 37°C for the wild type. In cOnTrast, tEmperAtures up to 45°C (FiG. 2C) had only a SMALl effect On PSi ElECtron transport In eitHer wild-typE Or mutant ThylaKoids. ConTrol thylaKOIds maintAinEd iN thE daRK AnD cold prior to aSSAy reTaIned higH raTes of PSiI eLecTroN trAnSport throUghout thE eXpErImEnt And foR Several hOuRs aFtErwArds.

THE disinTegraTion Of ThE ProTein comPLeX REspoNsIbLe foR oxYgEn evoLutiON haS been prOposed to bE thE Main CaUsE of irreVersible damagE oF PSII (Nash eT aL., 1985). To Test foR INhibitioN of the O2-evolving complex, dETached lEavEs froM wilD-type and mUtaNt planTs wERe firsT incubAted aT 40°C, For 15 OR 40 Min, reSPEcTivElY, to obtain a SIMilAr halF iNhibItion of fv/Fm (see Fig. 1A). Then, leaVEs wEre infiltrateD eiTher WITh HydROxYLamInE SolUTIons or with water As a control AnD fv/fm was measuREd aGaIn. HydroXylaminE is an ARtificiAl electroN donor thaT bYpasSES anY inhibitioN of the O2-eV
°C in the light did not re duce ØII b elow90%  of  t he s tart ing value in l e aves  of either the wild ty pe or  t h e mu t an t. In  contra s t, b oth  w il d-t yp e  a nd fa d3- 2 fad7- 2 fad8 mut ant  p hotosyntheti c  c apabilitie s,measured asFv/ Fm, si gn ifi c antly  di minis hed wh e n leav es were i nc u bateda t 40°Ci n  t he d ark. However, the mu t ant toleratedhigh t em p er a t ure s b etter than  t he wi l d type. To o b tai n  a 50% decrea se in Fv/Fm ,  it  was n ec ess a ry toincub at e  wi ld-type lea vesat 40°C f or onl y  15 min whereas  leave s f rom  the mu ta ntre q uir e d40m inof heattr ea tment  tor e a c h th e s amelevel . These resul tsindi c ate  that , intheda rk, h igh te mpera tu res mainly affe ct P SII activ ity  ( Nas het al . , 1985 ; S har key and  Zhang, 201 0; T o th  et al., 2011).

T oc o nf irm that  the p r im ar y  site of  h eat  dam a g e inv olve d  P SII, thy lakoid s  f ro m wild- ty pe and  m uta ntplant s  wer e isol ated and  incu b ated in the da r k for 10 mina tv a ri o us t emp eratures (2 5–45 ° C) b efor e  m eas u ringoxyge ne vo l ution at 25°C (Fig.  2 A-C).Bothwhole-chain ( Fig. 2A) a n d PSII-dep ende n te lectron transp ort ( Fig. 2B) w e re marke dly m ore tole rant to e l e vated te mpe rat ure  in t he  mutant compa r e d wi th  the wi ldtype. T hetem per atu re  resultin g in hal fin ac ti vat ion o f  PSII wa s42° Cfor  muta n t thyl akoid s co mp ar e d w ith 37° C  f o r  the  w il d ty pe.  I n con tras t , t emperat ures up t o 4 5 °C ( Fi g.  2C) ha d only a smal leffect onPS I e lectro n transpor t in either wild-type o r  mutant  th ylako ids.  Controlthy lakoid s m a intain ed inthe d ar k a n d  cold p ri orto  assay ret a i ned  high  r ates  of PSI I electron transpo r t t hroughout the  ex peri m e nt  an d  f o r s ev e ral h ours afterwards .

The dis in t eg ration oft hepr otein c omplexrespo n sible f or oxygen  evolutio nhasb e enproposed t o be the  main cau s e ofi rr evers ibl e dama ge  of  PSII  (Nash etal.,1985).  T o test  forin hibition  of the O2-evolving com plex,detac hed  leaves f rom wil d-type an d mu tant plant s w ere  firs t i n cubat ed a t  4 0°C ,  for15 o r  40 min,r es pec t i ve ly, to obta i n a s imila r h a lf inh ibit ion of Fv/Fm (see Fig. 1A). Then , le a v eswer e  inf il trated eitherwit hh y droxylam in e solutions  or with  w a ter a s a co ntroland Fv/ F m  w a s meas ured  ag ain. Hydr oxy la m ine isan  a r tifici al e le ctrondonort hatb y passes any inhib ition o f the O2- ev
°C in_the light_did not reduce ØII_below 90%_of_the starting_value_in leaves of_either the wild_type or the mutant._In contrast, both_wild-type_and fad3-2 fad7-2 fad8 mutant photosynthetic capabilities, measured as Fv/Fm, significantly diminished when leaves_were_incubated at_40°C_in_the dark. However, the mutant_tolerated high temperatures better than_the wild_type. To obtain a 50% decrease in Fv/Fm,_it_was necessary to_incubate wild-type leaves at 40°C for only 15 min_whereas leaves from the mutant required_40 min of_heat_treatment_to reach the same_level. These results indicate that, in_the dark, high temperatures mainly affect_PSII activity (Nash et al., 1985; Sharkey_and Zhang, 2010; Toth et al.,_2011).

To confirm that the primary_site of_heat damage involved PSII, thylakoids_from wild-type and_mutant plants_were isolated and_incubated in the dark for 10_min at various_temperatures (25–45°C) before measuring oxygen evolution_at_25°C (Fig. 2A-C)._Both_whole-chain_(Fig. 2A)_and PSII-dependent electron_transport_(Fig. 2B)_were_markedly more tolerant to elevated temperature_in_the mutant compared with the wild type._The temperature resulting in_half_inactivation of PSII was_42°C for mutant thylakoids compared_with 37°C for the wild type._In contrast,_temperatures up_to 45°C (Fig. 2C) had only a small effect on PSI_electron transport in either wild-type or_mutant thylakoids. Control thylakoids_maintained in_the_dark and cold_prior_to assay_retained high rates of PSII electron transport_throughout the_experiment and for several hours afterwards.

The_disintegration of the protein_complex_responsible for oxygen evolution has been_proposed to be the main cause_of irreversible damage of PSII_(Nash_et_al., 1985). To test for_inhibition of the O2-evolving complex, detached_leaves from wild-type_and mutant plants were first incubated at_40°C,_for 15 or 40 min, respectively,_to_obtain a similar half inhibition of_Fv/Fm_(see_Fig. 1A). Then, leaves were_infiltrated either with hydroxylamine solutions or_with water as a control and Fv/Fm was measured_again. Hydroxylamine is_an artificial electron donor that_bypasses_any_inhibition of the O2-ev