Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. Data may be preliminary. tesi w 4seiswt ieetsl tolerance salt Essemine different Jemaa with species salt C4 under two activity in oxidase stress terminal plastid of responses Contrasting aelwPO ocnrtos(bu TXpoenpr10PI;Lno ta. 03;i contrast, temperatures, in high as 2003); such al., stresses et abiotic Lennon to conditions PSII; exposed non-stress 100 plants under in light per response reported moderate protein in stress been in PTOX in involved have grown and 1 also levels Plants 2015), (about is PTOX 2011). al., elevated concentrations Wen, PTOX et PTOX (Nawrocki and 2001). at dark low Sun development al., located under have 2011; (Cournac plastid et potential is chlororespiration al., the redox 2012), PTOX Aluru et for chloroplast al., chloroplasts, 1999; (McDonald essential et poising In Trouillard is al., 2000), 2010; 2007). it al., et al., and al., et et (Carol (Okegawa 2003) et plants transport al., Shahbazi electron et in 1999; photosynthetic (Lennon biosynthesis the al., stroma carotenoid et in the Wu discovered and facing 1999; was lamellae al., stroma that et the oxido-reductase PTOX, Carol oxygen transfer, 1994, plastoquinol electron al., alternative plastid-localized & of in a immutans Stepien involved is so-called protein 2008; PTOX a Colmer, C protein reactive in that & The tolerance and report Flowers salt we ions, 2005; to study, related toxic Klobus, this be & of might In (Stepien compartmentation which systems 2009). biosynthesis, 2015), scavenging Johnson, osmolyte al., developmental, (ROS) plants et homeostasis, include Acosta-Motos how species that ion survive 2004; oxygen mechanisms of To al., the modulate complex Mechanisms 2008). et of Tester, with to (Taji & half adapt strategies serve 2004). Munns biochemical and about 2001; and Tyagi, respond and (Zhu physiological plants & study quality area morphological, intensive stress, (Sairam cultivated harvest under salt world’s are salinization and overcome stress the and productivity by salt of crop affected tolerate fifth and/or affects are respond one adversely lands Approximately that irrigated stress 2004). world’s environmental Schroeder, major & a (Horie is used salinity mechanism Soil major a is PTOX SA of INTRODUCTION for up-regulation times 1 the 3˜4 that stress. about suggest salt by SV these with increased for All cope PTOX not SV. to of but by SA for levels SA dropped species less protein for much C4 SA and activity flow or (PTOX) halotype mRNA electrons in oxidase by not the restrict activity terminal but to both its plastid stress however, (DBMIB) that enhanced NaCl showed Cytb6/f days; an under further of showed 12 NDH-dependent We we site for the O2, Qo-binding stress. NaCl 21% of the NaCl mM or activation of under 50 2% inhibitor an oxidation either to in and observed P700 at exposure (n-PG) mM, also increased PSI, inhibitor upon towards 100 We an and PTOX times showed than Using (SV) 2.4 change. SV higher 25%. about viridis no concentrations, level about by almost Setaria O2 NaCl SV showed different glycophyte to in SA Under a pathway exposure mM. while cyclic following species: 550 treatment, survive to C4 NaCl to two up following NaCl unable in level tolerate was stress could SV NaCl SA (SA). to contrast, alterniflora photosynthesis Spartina of halophyte responses a the reports study The Abstract 2020 5, May 1 Song Qingfeng hnhiIsiue o ilgclSine hns cdm fSciences of Academy Chinese Sciences Biological for Institutes Shanghai 1 eynChen Genyun , 1 igJ m Lyu Amy Ming-Ju , rbdpi thaliana Arabidopsis 1 n i-un Zhu Xin-Guang and , 4 plants. hc hw aigtdla hntp Ree,16;Wte et Wetzel (R´edei, 1963; phenotype leaf variegated a shows which 1 iga Qu Mingnan , 1 1 1 hha Perveen Shahnaz , 1 aedKhan Naveed , 1 , Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. Data may be preliminary. hr Dsad o pia est.Terslso h h otn eeepesda gprga fresh gram parameters per II mg photosystem as of expressed Assessment were content 2.5 (1989). Chl al. was the et content of Porra Chl results by Total given The (mg CA). g density. Chl Creek, using (mg optical total nm Walnut weight for 645 equation: Inc., stands and 4 Varian following 663 OD Varian, at the at where with monitored Bio to washed was (80%) (50 absorbance first according acetone their were spectrophotometer calculated subsequently ml g) visible and (0.1 min 1 UV segments 5 a in for Leaf g (1989). kept 13,000 al. at then et centrifuged and Porra to water according distilled determined was content Chl measurements content (Chl) Chlorophyll 2.4 F). 900 AAS PE HNO of ml the 10 K 105 with of at extracted first composition were dried samples º The Powdered were analyses. samples nutrient leaf days. mineral The for 15 water. 70 to deionized at with up subsequently washed for and harvested NaCl were Leaves mM (Na cations 550 (1950). monovalent and Arnon of and Determination 400, Hoagland washed 2.3 by 250, were described roots 100, Four-week-old as transfer, was 50, water. plants medium 0, deionized the hydroponic with with During rinsed treated solution. then were water hydroponic tap to with applied adequately was treatment for (NaCl) medium Salt hydroponic treatment to (NaCl) till them soil stress transferring the Salt on before 2.2 plants daily water growing spraying healthy treatment. by stress ensure wet salt to maintained routinely was For added which treatment. was soil, salt germinated. for potted seeds system wet hydroponic the into a sown to directly transferred were where leaves expanded large with 120 of - seedlings 80 young of the PPFD When soil. potted into containing year. transferred one were about they for germination, plastic SA viable After sealed remain germinate. in they till (cloth) and water tissue 1992) wet McIninch, in & of (Garbisch stored Seeds dormancy were break spikelets to cleaned (stratification) The 2016. and 4 2015 at in November of end of Seeds Material Plant stress. 2.1 salt under METHODS photosynthetic species of AND this maintenance MATERIALS the of 2 for efficiency halophyte role photosynthetic critical the high a species how hence played glycophyte have of and might a physiology mechanism which PTOX, to potential of compared understand 2013). expression increased that al., to high et show effort and (Laureau we an temperatures light stress, low in UV 2009), study, and Johnson, this 2012) & al., In (Stepien et (Ivanov salinity light high visible 2006), of (Quiles, intensities drought and light high .Tersligsltoswr lee hog hta le ae,dltdadaaye o Na for analyzed and diluted paper, filter Whatman through filtered were solutions resulting The C. + aincnetain eedtrie iha tmcasrto pcrpooee (PerkinElmer, spectrophotometer absorption atomic an with determined were concentrations Cation . eeaot2c nlnt n tre reig hywr eoe rmtegasptidse.Trays dishes. petri glass the from removed were they greening, started and length in cm 2 about were ° nterefrigerator. the in C SV priaalterniflora Spartina SA eerne eea ie ihtpwtradte rnfre oPtidse n oee with covered and Petri-dishes to transferred then and water tap with times several rinsed were -1 edig eekp nora eprtr ewe 25˜27 between temperature a at indoor kept were seedlings rs egt(W)adcluae ae nteetnto offiinsadteequations the and coefficients extinction the on based calculated and (FW)) weight fresh μ ° o m mol o 2h n hnwihe o r egt ypiie evswr ildt powder to milled were leaves Lyophilized weight. dry for weighted then and h, 72 for C SV -2 SA rwudrtesm htpro n eprtr odtosas conditions temperature and photoperiod same the under grew s -1 auesesrqietot he ots fe-ieig e trg ncold in storage wet after-ripening, months, three to two require seeds mature ( ihapooeido 68hu lgtdr) w-ot l elh plants healthy old Two-month (light/dark). hour 16/8 of photoperiod a with SA eecletdfrom collected were ) + 2 n K and San-San SV ° + ne ihsl tes(0 mM), (500 stress salt high under , o n otowes hnsmlswere samples Then weeks. two to one for C content ) · L -1 aei ot atSaga iya the at city Shanghai East South in Lake 80 OD x (8.02 = ) ° ,udrfloecn ih ta at light fluorescent under C, SV n 10-week-old and 3 663 01)fr6 i t95 at min 60 for (0.1N) 2.1xOD x (20.21 + ) SA oeaehg salt high tolerate SV SA SA r seeds dry , ° Nutrient . o h, 2 for C SA showed plants + 645 and ), Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. Data may be preliminary. efsmlswr okdi MNTslto otiig5 Msdu hsht p .)fr1 h the that, 12 After for darkness. 7.5) under (pH h 12 phosphate for sodium 3.8) (pH mM MES 50 (H mM containing peroxide 10 (O containing solution hydrogen radical solution NBT detect (DAB) free mM To diaminobenzidine superoxide 6 For darkness. vacuum-infiltrated in NBT). first soaked under without were were leaves or Detached samples (with per- modifications. was leaf solution minor (NBT) appropriate with tetrazolium their (2009) nitroblue with in al. staining et histochemical Dong (ROS) following (ROS), species formed conductance species oxygen oxygen the reactive reactive of 2009). detect of Johnson, measure To localization & a well Stepien histochemical as fitted 2003; taken situ was Johnson, was In and & This 2.9 reaction (Golding first-order chain constant. decay a in absorbance transport rate The to rise electron a closely rapid shown). the yielding (not approximated of a curve, flash salt) induced mono-exponential the or flash a of (ctrl a duration with conditions of be the such during to application under decrease found conditions, no was curve these intensity with Under of This signal, PPFD absorbance used. 2008). a the conditions Schreiber, at & all was SP Klughammer across pulse width 1994; saturating saturating 100-ms Schreiber, a The & Notably, (Klughammer modifications. AL. darkness slight of with termination PSI the with of 8000 simultaneously state darkess redox under the given monitoring chain section transport electron previous the the of (g conductance chain the estimate transport To electron the of Conductance 2.8 1804 P to nm. 0 875 photosynthesis minus state 830 steady P at during of changes PSI state through redox flow the electron estimated photosynthetic we experiments the monitor for to setting order The In (GDS). station P and distribution of 2 gases Evaluation (e.g. a 2.7 concentrations by different CO supplied with of sources are levels Oxygen study) different CO chamber. this using control the in was to to used source which analyzer as the gas fluorimeter 21% from infrared oxygen Dual-PAM-100 of portable the flow 6400 the of Li-COR detector-emitter the 2000 to the or hole on a mounted CO through of tightly connected control CO precise was control the enable chamber to to to developed This analyzer and according custom-designed gas was curves chamber Infrared OJIP special with A original together the PAM from of calculated Setting were 2.6 2004) S1 al., Table et the (Strasser in JIP-test listed so-called parameters F the intensity). All light actinic negligible of one beam least at Chl saturating minimum for F to dark-adapted PSII. exposed were of were efficiency Plants leaves PSII expanded F of fully (2017). level evaluation and fluorescence (5,000 healthy the light al. actinic Then, for et nm) (625 measurements. UK) Essemine orange-red before by Norfolk, 25degC details Lynn, at in King hour Hansatech, described (M-PEA; as analyser parameters Chl efficiency the plant using tional assessed was efficiency PSII sstrto us S)o PDo 8000 2008) of al., PPFD et of Dalcorso (SP) 2008; pulse Schreiber, & saturation P Klughammer ms the 2005; of in al., dependence et examined light Zygadlo was 1994; The Schreiber, 2008). & Schreiber, ( Klughammer & photochemistry Klughammer PSI 2005; favor al., to illumination far-red μ o m mol μ L L μ o m mol -1 -2 yL-o n i nte idwt noye oreeupe iha xmtrt adjust to oxymeter an with equipped source oxygen an to window another via and Li-cor by ) s a -1 SV ursec teitniyo Chl of intensity (the fluorescence -2 a ple n h ea nasrac olwduo rniinfo h 0 sS to SP ms 100 the from transition upon followed absorbance in decay the and applied was s v -1 m (F and ( 700 Plvl ersnstemxmmyedo Chl of yield maximum the represents (P-level) m Δ -F )te eue ntedr n nlyoiie oamxmmlvlo P of level maximum a to oxidized finally and dark the in reduced then A) SA eo tt nlae fS n SA and SV of leaves in state redox 0 2 omxmlfloecnelvlF level fluorescence maximal to ) n O and lns h a-e ih F)itniyue a 102 was used intensity (FR) light Far-red The plants. 700 700 2 a xdzdt P to oxidized was ntelgtb esrn xdto fP of oxidation measuring by light the in a sdpce nFgr n ie nsplmna data. supplemental in video and 1 Figure in depicted as was μ o m mol a v μ /F o m mol ursec nuto F)tcnqe eue h multifunc- the used We technique. (FI) induction fluorescence -2 0 aaee ersnstefntoa ecincne fPSII. of center reaction functional the represents parameter s -1 2 a -2 Δ rvddb h E o eod h ai fvariable of ratio The second. 1 for LED the by provided ) O s 700 ursec fdr-dpe apewt measuring a with sample dark-adapted of fluorescence 3 mx lgamradShebr 94 yal et Zygadlo 1994; Schreiber, and Klughammer Amax; -1 2 ,dtce evswr mesdi Mo ,3’ 3, of mM 5 in immersed were leaves detached ), + a ple ne akrudA n FR. and AL background under applied was tdffrn neste fatnclgtrnigfrom ranging light actinic of intensities different at ETC (g m ETC (F ) v ) /F eue iia xeietstigto setting experiment similar a used we , m a a sdt vlaetemaximum the evaluate to used was ) ursec n F and fluorescence 700 700 xdto ai ( ratio oxidation ihntela sabsorbance as leaf the within 2 n O and μ 2 o m mol - n O and 2 . characterization, ) 2 0 -2 2 supply Olvl sthe is (O-level) 2 environments. Δ s upis(390 supplies -1 A/ 700 n 100 a and Δ nvivo in + Amax; under , Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. Data may be preliminary. opn codn h eunehmlg ewe pce hc a 3 sebatsqecsalignment sequences blast (see 63% was which ( data). species species supplemental between both in homology et for results Mudd sequence PTOX in the against described according raised as performed company antibodies was Polyclonal Immunoblotting (2008). Laboratories). (w/v) (Bio-Rad al. 4% kit assay glycerol, protein 20% Bio-Rad 6.8, a pH (1984). Tris-HCl, Plesnicar mM & (v/v) 125 Cerovic 5% using in SDS, membranes from described extracted as were isolated proteins were Thylakoids thylakoids analysis, (western-blot) immunoblot For analysis Western-blot 2.12 and fresh. control prepared were of of solutions leaves solutions inhibitor the stock All The transport, 50 electron Sigma). or p-benzoquinone, PSII Sigma) overall ester; to acid-n-propyl PTOX 3,4,5-trihydroxy-benzoic the SA of in salt-treated contribution transport the electron determine in To contribution PTOX analysis. the of for Detection calculated determined 2.11 was were tubulin-alpha replicates gene technical housekeeping and against biological gene analysis complete of qPCR website expression for Relative 2 Information used S2. as: Biotechnology Tubulin-alpha Table Biosystems and for in Applied PTOX Center listed StepOnePlus, for National were primers (ABI the The System of https://www.ncbi.nlm.nih.gov). PCR Primer-Blast (NCBI; Real-Time using 95 a designed parameters: were on with cycling qRT-PCR USA) following template Biosystems, the a (Applied with as Mix USA), Master lco., cDNA PCR http:// Quan- strand Green Technologies, SYBR fist invitrogen.com). using Life the http://www. performed Technologies, (Invitrogen was Life PCR (Invitrogen kit real-time Kit titative Purification Synthesis RNA cDNA using and VILO Plus CA, measured species (1 SuperScript TRIzol was both microgram Carlsbad, using from One sample (Invitrogen, sampled extracted RNA www.invitrogen.com). Kit were was each Mini Leaves RNA of Technologies). RNA total Concentration (NanoDrop Purelink spectrophotometer using instructions. 2000 leaves manufacturer’s NanoDrop mature to from according extracted USA) qRT-PCR. was for in RNA (Tub gene gene Total reference alpha housekeeping as Tubulin each ? in of Tub sequences index selected the similarity therefore between highest index the 2013). similarity al., obtained the (18S), et on rRNA Kumar (APRT; Based 18S transferase on viz., 2012a). phosphoribosyl study adenine are: recent II), genes a POL These In (RNA (Tub II tubulin 2013). polymerase reference alpha al., RNA appropriate (Act2), et (TLF), actin2 an (Kumar (EF-1a), select families factor-1a gene to elongation and screened classes were functional 2013) different al., et for et (Kumar gene Zulfugarov genes till by housekeeping ethanol analysis candidate described 50% qRT-PCR Eight as in and preserved (99.5%) purification were alcohol for extraction, Chl water in of RNA in devoid incubated 2.10 leaves re-boiled were Afterwards, were leaves leaves Chl. Then (NBT and of solutions removal solutions each). photographed. respective complete these min their by till discarded in (2014) (1 were (1:1:4 leaves solutions times al. lacto-glycerol-ethanol boiling the 3 by with then imaging leaves to min to the 2 2 prior soaking leaves for the by DAB) from or stopped removed (H were was peroxide Chl NBT) O hydrogen vol). and formed generated (DAB and the (500 reactions DAB light and Both between high NBT interaction moderately between the to interaction reflect exposed the was reveal leaf spots the of surface adaxial -CT ( SA Δ T=C,gn fitrs-T uui-lh) sdsrbdb ia cmtgn(01.Six (2001). Schmittgen & Livak by described as Tubulin-alpha), interest-CT, of gene CT, = CT SV and β- and ecpotao,01 wv rmpeo le rti ocnrto a siae using estimated was concentration Protein blue. bromophenol (w/v) 0.1% mercaptoethanol, SV. SA priaalterniflora Spartina hs ih ee aebe eotdon reported been have genes eight These eevcu nlrtdwt ihrwtro ih5mM 5 with or water either with infiltrated vacuum were μ uui a sda oskeiggn KrnadSubudhi, and (Karan gene housekeeping a as used was tubulin , )o oa N a sdt yteiefis tadcN with cDNA strand first synthesize to used was RNA total of g) n P eepeae nehnladDMBi methanol. in DBMIB and ethanol in prepared were -PG ° 4 o 0s 55 s, 10 for C μ BI (2,5-dibromo-3-methyl-6-isopropyl- DBMIB M α eai italica Setaria ,wihi rud8% nti td,we study, this In 85%. around is which ), α - ,bt uui (Tub tubulin beta ), 2 ° . o 0s n 72 and s, 20 for C oee,tebonsoso h leaf the on spots brown the however, ; μ 2 o m mol SA O 2 ttepeec fperoxidase. of presence the at ) and FxalMle) representing Millet), (Foxtail -2 SV s -1 n o h h dark-blue The 1h. for ) ° poy alt (n-PG, gallate -propyl eedsge ythe by designed were ) o 0s rmr for Primers s. 20 for C β ,tasainfactor translation ), SV and SA we , Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. Data may be preliminary. otn Fgr A;tettlClcnetainatr1 aso attetetwt 0ad10NaCl 100 and 50 with treatment salt of of treatment days contrast, 12 In after respectively. concentration 58%, and Chl 42% total by the dropped 4A); (Figure content NPQ untreated untreated components: in in decay content quenching Chl non-photochemical total and The leaf 3E-F). in (Figure content mM Chl 250 3.3 than higher for concentrations k NaCl ratio ratio at this the especially in by decline reflected dramatic also K This a 3D). the (Figure treatments, concentration of NaCl days 12 in after However, in NaCl. 30% mM about 100 by higher K was the medium treatment, salt-free al., with et watered plants (Barhoumi of accumulation salt following size SA cell performed in study increase Earlier an vacuole. to due the 2007). of mainly barrier in was second salt thickness a of epidermal represents sequestration mechanism the exclusion halophyte This besides on concentrations Spartina. NaCl for surface high leaf against the to NaCl than of leaf of the that in to NaCl similar nearly Na was The ). Na (Figure leaf Na whereas 3A), . (Figure and 0 between concentrations NaCl Na Na of of concentration accumulation The mortality. in considerable for in higher used result not not were did treatments to concentration NaCl higher so treatments of NaCl Exposure following black leaf 2E-F, of in (Figure Plants sequestration exposure potassium days’ 15 and green and Sodium and 10 3.2 red after 2B, mM) of to (Figure (550 mM PSII exposure 100 concentration Therefore, of or NaCl spider). days 50 high either 5 for at after only salt observable that these to exposure observe in 2004). for of in we However, days al., PSII change Herein, spiders). 10 et apparent on after showed concentrations. Strasser pronounced NaCl parameters more NaCl S1; became of PSII Table different NaCl, to effects 1, mM F) (Fig the 100 (C, JIP-test detail days the in 15 using from study and evaluated parameters To was PSII the JIP-test mM). Therefore, the The evaluated (50 NaCl. we without curves concentration species, control induction NaCl for two the OJIP low affected to of relatively compared not J-test concentrations was at NaCl the PSII mM) and for of (550 shown) that, function high in not show and parameters (data experiments mM) However, PSII level (250 treatment of Fo shown). stress most the not on Salt increased (data stress salt curve. mM) of (50 OJIP effect concentration the the unravel i.e. to curve, 2004) al., et (Strasser both JIP-test test a JIP used and We induction fluorescence a Chlorophyll 3.1 RESULTS 3 and SV SV SA and SV iee loi hi K their in also differed SV hnin than SV SA euou littoralis Aeluropus and oNC ocnrtoshge hnrsle npatdahbfr h n fteexperiment; the of end the before death plant in resulted than higher concentrations NaCl to + eut eitdi iue2wr eie rmtefs hs Chl phase fast the from derived were 2 Figure in depicted Results . evs(iue4-) xoueof Exposure 4A-B). (Figure leaves SA otn ftela in leaf the of content + + SV ntela of leaf the in eesmaue fe 2dy alteteti priaepsdt 0 n NaCl and 400 to exposed Spartina in treatment NaCl days 12 after measured levels eegonfr4o ek eoeterepsr oarneo atconcentrations. salt of range a to exposure their before weeks 8 or 4 for grown were SA SA Fgr AB.Ti ieec iapae fe xouet at u oaquick a to due salt, to exposure after disappeared difference This 3A-B). (Figure h eito ntePI aaeescluae ihJPts a uhls and less much was JIP-test with calculated parameters PSII the in deviation the , ne l atcnetainrne(0-5 M.Ti soigt h exclusion the to owing is This mM). (100-550 range concentration salt all under SV SA + SA SV a oesniiet atsrs,a oprdto compared as stress, salt to sensitive more was otn in content hr a niiilices nK in increase initial an was there , hwdn/rsih ieec nteOI nuto uvsfrmoderate for curves induction OJIP the in difference slight no/or showed ujce ool al(iue3-) tNaCl, At 3A-B). (Figure NaCl only to subjected pce htcntlrt pt al hwdta nices nleaf in increase an that showed NaCl, to up tolerate can that species a , SV SV + + ocnrtosi h ef een h ocnrto fK of concentration the Herein, leaf. the in concentrations SV SA ocnrto ntela rdal erae iha nraei the in increase an with decreased gradually leaf the in concentration SA h cuuaino Na of accumulation The . A n )and C) and B (A, SV evsudrsl ramn a rud45tmshge hnthat than higher times 4.5 around was treatment salt under leaves u eyls n otymiti tbewt iecus in course time with stable maintain mostly and less very but + oee,i a togyihbtdado aae for damaged and/or inhibited strongly was it however, ; SA cuuainicesdsapyin sharply increased accumulation erae osdrby seilyatr4ad8dy ramn at treatment days 8 and 4 after especially considerably, decreased nrae es vna ihretra ocnrtoso NaCl of concentrations external higher at even less, increased SV 5 SA oNC eutdi rgesv eraei Chl in decrease progressive a in resulted NaCl to SV D n )epsdfr5(,D,1 B E) (B, 10 D), (A, 5 for exposed F) and E (D, SV + xoueof Exposure . + Fgr A re pdr.Tesl effect salt The spider). green 2A, (Figure nlae of leaves in + SA /Na ihteices nNC concentration; NaCl in increase the with + SA ncnrlla isewsconsiderably was tissue leaf control in ih5 n 0 alddntresult not did NaCl 100 and 50 with + evs(iue3-) olwn salt Following 3C-D). (Figure leaves Fgr EF,weew observed we where 3E-F), (Figure SA SV SA SA SV a uhlwra external at lower much was evsoe h experiment the over leaves oNC ocnrto up concentration NaCl to a . SV vnmdrt NaCl moderate even , ursec induction fluorescence cuuae more accumulated + nla tissue leaf in at slow fast, SA SV defense SA even , Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. Data may be preliminary. h TIflostesm rn n erae ytesm mut(iue5G-H). (Figure amount same the by (g decreased ETC and the SV trend of same P conductance the oxidized follows the of ETRI plants, P the proportion untreated irradiance, the control Despite in increasing 5C-D). concentration With (P (Figure PSI species of plants. donor both electron control primary irradiances. the in saturating of concentration at state ETR redox the PSII of of Measurements decrease a 400 in resulted around (2%) at oxygen maximum 1806 its to reached 0 (AL, species these levels of performed. irradiance relevance the of was assess range (2000 flow To a electron 2013). at al., of Light including et Actinic oxygen, dependence Shirao involving 2004; oxygen reactions al., the the et are pathways, (Chen sinks reaction known Mehler commonly and most photorespiration The C generation. both NADPH in support H by salt generated under electron oxygen The molecular to flow Electrons 3.4 in photoinhibition NPQ of of total NPQ occurrence majority (Figure in in the increase quenching the suggesting The high-energy-state that (NPQs), 4C-D). was conservative showed In (Figure it more quenching quencas decay. of that the NPQ leaves dark indicating of in of NPQ (NPQf), part of components dark a while phase the relaxing 4C-D); taken each in slow were of rapidly and for MeasurementNPQ relaxed magnitude treatments fast quenching the NaCl the 2009). quantify kinetics mM of al., to relaxation 400 quantification darmeasured et the and under Johnson in 100 recovery 2000; differ to NPQ Johnson, which daosure & otoinhibition, 16 illuminationell or after light quenching high-energy-state actinic protective after a NaCl either at of e.g. , 4B). than (Figure higher SA content concentrations Chl NaCl total at in In except decrease content ˜20% Chl a total as the there in treatment, decline significant any in ntecnrl lcrntasottruhPI erae lgtyudrlwO low under slightly decreased PSII through transport electron control, the in erae(bu 5)i h D ciiy(iue6.Ti svr ieyatiual oteatvto of activation the in to declines attributable PIR likely of very activity is the This Hence, 6). stress. (Figure salt activity under NDH Spartina the in PTOX in 25%) (about decrease of for leaves However, in in PIR activity 6). NDH of NDH the magnitudes in higher The increase times (2016). two an than al. F more et of observe Essemine (PIR) we light rise actinic post-illumination off the switching as and after assessed monitored was was PSI cence around activity pathway cyclic species NDH both in flow stress electron cyclic salt (NDH)-dependent under dehydrogenase NAD(P)H of O Activity low 3.5 at decreased mM) (250 salt P in Conversely, of in 5G). presence (Figure or FRQ the across absence in cyclization the decline via negligible potentially a PSI to through flow by accompanied is P This oxidized 5C). O of low under proportion revoked The ETR completely the was in PSII increase through an transfer in electron resulted NaCl to exposure SV talNC ocnrtos(iue4-) h P nraein increase NPQ The 4C-D). (Figure concentrations NaCl all at 700 xoe o10m alsoe oe ETR lower showed NaCl mM 100 to exposed SA μ altetetrsle na nraeo P,wieNQrmie iia rogtyicesdin increased oightly or similar remained NPQ while NPQ, of increase an in resulted treatment NaCl , L L f xdto n ali g in fall a and oxidation ne ohcnrladsl tescniin eedslydi iue6 ne omlconditions, normal Under 6. Figure in displayed were conditions stress salt and control both under poortcinprocess). (photoprotection -1 SV n ihr2%o %o O of 2% or 21% either and ) SA u ohg al ohfrso P unhn nrae nrsos osl treatment salt to response in increased quenching NPQ of forms Both NaCl. high to due lnsepsdfrtesm iepro 1 as o20m alehbtdasignificant a exhibited NaCl mM 250 to days) (12 period time same the for exposed plants 2 pitn a eue yatraiesns nadto otecmo ikto sink common the to addition in sinks, alternative by used be can splitting O 700 ETC (P 700 SV Fgr 5D-F). (Figure + nsalt-treated in ) lnsedrd5 MNC o 2dy yaot23 ie (Figure times 2.36 about by days 12 for NaCl mM 50 endured plants 2 eadestedge fNC ramns h ERT the treatments, NaCl of degree the Regardless . μ SA o m mol a oprtvl esadwsmil u oa increase an to due mainly was and less comparatively was II -2 II thg CO high at 6 in s SA -1 SV g SA Fgr AB.Epsr fcnrlpat olow to plants control of Exposure 5A-B). (Figure μ T,rsle nasih nraei h electron the in increase slight a in resulted ETC, hnin than SV o - -)a h rsneo auaigCO saturating of presence the at s-1) m-2 mol 2 a infiatyhge ne o O low under higher significantly was oprdt nrae lns hsices in increase this plants; untreated to compared Fgr H.Ti asdb nenhancement an by caused This 5H). (Figure and 2 700 700 hntecnrlpat Fgr A.As 5A). (Figure plants control the than SV SV 2 eaeporsieymr xdzdin oxidized more progressively became SV 4 (P SA ocnrto Fgr 5B). (Figure concentration ih ersle rmamodulation a from resulted be might species Fgr ) h eut hwa well as show results The 6). (Figure 700 700 and epciey(iue4-) The 4C-D). (Figure respectively , + eeldsih ffcso oxygen of effects slight revealed ) ETC a nestv oteoxygen the to insensitive was ) SA SA iue5-)dcie and declined 5E-F) Figure ; nfvu fta fPTOX. of that of favour in 2 eesbetdt different to subjected were Fgr A.I contrast, In 5A). (Figure SA S T (ETR ETR PSI , o h fluores- Chl II 2 nboth in (Figure SV I in ) SV 2 , Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. Data may be preliminary. Fgr B,fligteeyt h oto ee ree lgtylwr(iue7) neetnl,a o O low at Interestingly, F 7B). the (Figure in lower slightly decrease even a or observed level (O we control mM plants, the 5 salt-stressed to with in thereby infiltrated leaves falling in 7B), 45%, (Figure and control In 32 for 2015). about case al., by the et also Nawrocki to was 2013; insensitive This al., was 7A). et (Figure Shirao not 2012; or al., treatment et (F Trouillard yield 2011; Wen, quantum inhibitor & PTOX Sun the 2011; of al., solution a salt-treated acid- or and O control water to from either PSII from obtained with transfer leaves electron least might on in 2009) the at role performed a al., oxidoreductase, or play were et may quinone-oxygen reason, PTOX Heyno putative the the whether 2007; the determine unlikely for To al., that well is et possibility as this (Shahbazi role the protein that a test IMMUTANS suggests played or we have oxygen (PTOX) here to oxidase So terminal parameters plastid reason. PSI only of the sensitivity not a the of lack at O the spartina of in photo-reduction the treatment Usually, (O salt oxygen molecular under O CO to 21% saturating PSII directly with transfer control of electron either turnover to to attributed compared additional oxygen, the the 21% in of and/or protein presence PTOX efficiency plastohydroquinone:oxygenoxidoreductase of improved a level as The high (PTOX) the oxidase by terminal oxidation Plastid (2012), pool 3.6 PQ from al. of PTOX et activity overexpressing Ahmad enhanced tobacco of line. the the in that over-expressed to for PIR with valve attributed line the safety is alternative in a in PIR an as are decrease represent function findings a may may our reinhardtii shown PTOX it regard, have Thereby, far, this PSII). authors So In and where PTOX (PSI chain. by 8). photosystems transport pool both and of photosynthetic for to PQ protection 6 NDH) the damages the for avoid and of (Figs. routes or (PTOX oxidation linear cyclic pathways and the NDH cyclic two Eventually, to by these pathway respectively. between re-reduction pool, competition its PQ efficient overcomes the an of of oxidation/reduction existence the the reflects This lcrntasot omaueeeto o ooye xldn n otiuino h elrreaction, Mehler the of contribution any b excluding cytochrome oxygen the to with flow infiltrated electron were measure leaves To transport. in electron membrane the of side of effect The h lcae(rsotg)i h lcrn o oad S u otepeec fDMBadtesecond to the of due and extracts limitation DBMIB membrane of First presence thylakoid PSII. O the of the beyond to in analyses flux due drop Western-blot PSI electrons the towards the to flow linked in electrons tightly restriction the is in double curtailment shortage) a (or as blockage explained the be might F 7D) in decline dramatic The of F of inhibition SA F inhibited strongly In cytb also 1999). the al., beyond et flow Schoepp electron 1991; Q the al., of et inhibitor Rich specific a (DBMIB), 3-methyl-6-isopropylbenzoquinone n 2 a c satria lcrnacpo yoiiigtepatqio (PQH2). plastoquinol the oxidizing by acceptor electron terminal a as act may ) P-estv lcrntasoti h aelae Fgr BadD). and 7B (Figure leaves same the in transport electron -PG-sensitive evs BI nyprilyihbtdF inhibited partially only DBMIB leaves, n poy se;J¨te l,20;Jsee l,20;Knz 04 os ta. 06 oil-enset Houille-Vernes 2006; al., et Rosso 2004; Kuntz, 2003; al., et Josse 2002; al., Jo¨et et ester; -propyl C-TX oprdt T(ha ta. 02 n hydmntae httedces in decrease the that demonstrated they and 2012) al., et (Ahmad WT to compared (Cr-PTOX) n 2 PSII P ugssaptnilatvt fpatdtria xds PO)lctdo h stromal the on located (PTOX) oxidase terminal plastid of activity potential a suggests -PG ocnrto f2000 of concentration PSII n h xeto BI nestv,oye-estv F oxygen-sensitive insensitive, DBMIB of extent The . P Fgr B.F 7B). (Figure -PG a nestv to insensitive was ) SA PSII PSII huhti osntecueaptnilcnrbto fteMhe ecinto reaction Mehler the of contribution potential a exclude not does this though huharsda F residual a though , 6 SA f eadesteNC ramn Fgr C.I control In 7C). (Figure treatment NaCl the regardless /f, ntepeec fDMBa o O low at DBMIB of presence the in . 2 a cu tteacpo ieo S i h elrrato;however, reaction; Mehler the via PSI of side acceptor the at occur may μ L L PSII n SV -1 P,rgrls hte h lnshv enepsdt NaCl to exposed been have plants the whether regardless -PG, eecuetecnrbto fpooeprto oti effect. this to photorespiration of contribution the exclude we , esrd1 asatriiitn altetetwsreduced was treatment NaCl initiating after days 12 measured 6 f(Cytb /f hsams opeeyaoihdteF the abolished completely almost this , PSII 2 PSII ee (2%). level ihdcesn O decreasing with 7 n 6 n loeeto rnfrrmie.I salt-stressed In remained. transfer electron also and f niio irmtyounn r2,5-dibromo- or dibromothymoquinone inhibitor /f) SA PSII n P,i h rsneo 1ad2 O 2% and 21 of presence the in -PG, poy alt ( gallate -propyl gis nesse vrrdcinadminimize and over-reduction intersystem against SA hssget togyta oeua oxygen molecular that strongly suggests This . 2 .Sneeprmnswr odce ne a under conducted were experiments Since ). 2 and SA r20m alwt %O 2% with NaCl mM 250 or SA SV 2 In . nsl rae prialae (Figure leaves Spartina treated salt in and 2 sn nioisrie against raised antibodies using SA SV ocnrto eutn ngreater in resulting concentration o bnigst Mli,18,1982; 1981, (Malkin, site -binding PSII n xoe o20m al F NaCl, mM 250 to exposed hc eevcu infiltrated vacuum were which P;3,4,5-trihydroxy-benzoic -PG; eraewssmlrt that to similar was decrease 2 esrmnso ETR of measurements , PSII SA Chlamydomonas n hrb the thereby and evs DBMIB leaves, 2 2 svr likely very is , respectively , SV PSII , PSII Zea II 2 Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. Data may be preliminary. . atsrs nue prglto feeto o hog h TXatvt nSpartina in activity between PTOX difference PTOX. the huge of through role a flow physiological electron is general of the There in up-regulation discuss PTOX we induced of then stress up-regulation and the Salt stress, of 4.1 salt finding with major up the cope discuss to first mechanism we section, this In DISCUSSION 4 10C-D). the salt-treated (Figure increased or surface control dramatically either treatment on salt spots that SV brown suggesting visible 9D), no showed (Figure in (ROS) (DAB) days species 12 oxygen reactive for of salt production mM 50 to subjected etti,w xmndtePI htihbto olwn atsrs npeec of in O presence electrons in 21% stress of or salt sink 2 following major (Q photoinhibition a DBMIB PSII is the or PTOX examined pool we that PQ this, suggest test high inhibitors a using experiments and Second, limitation side acceptor to PSII compared strong as a to between similar leads This in Q 1999). over-reduction pronounced Strasser, significantly more was & curves a OJIP (Haldimann of and J-step the pool stress, PQ salt to exposure After for enhanced 6). activity in (Figure CEF control times respective NDH-dependent 2.36 by their the to enhanced stress, was in salt activity higher NDH no the was times treatment, there two when than more i.e. conditions, was growth normal under First, Na of salt, exclusion under Therefore, that PTOX. suggest through 2009). data in al., salt-tolerance our et in here involved (Oh mechanism abundance a be SOS1 also higher exhibit halophytes Mostly, Na K the 2014). in increase or maintenance in that found the (O on radical free spots (500 superoxide light dark-blue generated high of the and appearance NBT the between interaction shows (NBT) of tetrazolium C edge nitroblue in with salt staining was under Histochemical PTOX generation 8A-B). of species (Figure abundance amount oxygen transcript transcript same the Reactive PTOX Similarly, the 3.7 of by inset). treatment and level NaCl 8A expression (Figure under the control elevated the though also to inset), compared and times In 3˜4 8A 8B). (Figure (Figure abundance decreased PTOX insignificantly in the than in abundance increase protein higher showed mays feeto rmPIflwit TXdie reactions. driven PTOX into flow PSI from electron of in PTOX to O both low to under sensitive severely ? more even the and on effect little has rae ihol 0m alfr1 assosawdsra rsneo rw pt nteleaf the on spots brown of presence widespread a shows days 12 for NaCl mM 50 only with treated TXrvae h rsneo 5kabn nbt pce Fgr ) o nrae plants, untreated For 8). (Figure species both in band 35-kDa a of presence the revealed PTOX SA SA + 2 oeac sascae ihSS niotrlclzdt h oteiems(h ta. 2002). al., et (Shi epidermis root the to localized antiporter SOS1 with associated is tolerance evsepsdt 5 MNC o 2dy Fgr B.Ti akbu tiigrvasthe reveals staining dark-blue This 9B). (Figure days 12 for NaCl mM 250 to exposed leaves Fgr ) u eut eeldta h etito neetosflwtwrsPO ( PTOX towards flow electrons in restriction the that revealed results Our 7). (Figure SV SA, u o in not but o bnigst fCytb of site -binding μ SV. SA o m mol ihtm,ete tbeo nices nteK the in increase an or stable a either time, with SV oprdto compared and n hr utb ao oreo lcrnwihacp lcrnin electron accept which electron of source major a be must There oprdto compared P n O and -PG -2 SV, PSII SV s -1 ..teicdn ih nrywsntmr isptdi h omo etin heat of form the in dissipated more not was energy light incident the i.e. .Hwvr hs akbu pt eesra l vrtesraeof surface the over all spread were spots dark-blue these However, ). in ne atsrs.I at vnudrnnsl odto,teei proportion a is there condition, non-salt under even fact, In stress. salt under SA SV + 2 dt o aa.TeicesdJlvli nidctro oereduced more a of indicator an is level J increased The data). not (data /Na 1% iue7) hspoie neiec hta ffiinl operating efficiently an that evidence an provides This 7B). Figure (13%, SA SA Fgr A u infiatydcesd? decreased significantly but 7A) (Figure - 2 6 SV A niio)a topei CO atmospheric at inhibitor) f Fgr B.Ti eet htapooto feetosfo SIis PSII from electrons of proportion a that reflects This 7B). (Figure + utemr,w on htudrsl tes h ee fNQwas NPQ of level the stress, salt under that found we Furthermore, . hnin than piayeeto cetro SI cuuainudrsl stress salt under accumulation PSII) of acceptor electron (primary SA samjrtatascae ihsl oeac Saaa&Pottosin, & (Shabala tolerance salt with associated trait major a is SA and andicesdsl oeac hog nrae lcrnflow electron increased through tolerance salt increased gained SV ramn ih20m aleeae TXaudneby abundance PTOX elevated NaCl mM 250 with treatment , SA SV SV ntelte ( latter the In . SA. eadn hi hslgclrsos osl.Hr,we Here, salt. to response physological their regarding iial,hsohmclsann sn diaminobenzidine using staining histochemical Similarly, . ne omlcniin(iue6;hwvr fe NaCl after however, 6); (Figure condition normal under 8 nadto oti nw ehns fsl tolerance, salt of mechanism known this to addition In SV u erae yaot2%in 25% about by decreased but 4 SV species + /Na 2 ,sl ramnsrsle naslight a in resulted treatments salt ), SA - (390 2 . + evs(iue1AB.I contrast, In 10A-B). (Figure leaves olwn xouet moderately to exposure following ) a bevd(iue3-) This 3E-F). (Figure observed was μ L L PSII -1 in n CO SA SA P PO inhibitor) (PTOX -PG 2 SA n TXa new a as PTOX and n npeec of presence in and ) ne ohnormal both under SA ne atstress. salt under ne at To salt. under SA compared , SV + n-PG should leaves SA SA ) , Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. Data may be preliminary. hr a oaprn eraei E ne at(iue5) hc ugssta h htsse Iin II in photosystem ROS the of that accumulation suggests higher which much 5B); observed (Figure we In salt salt, 1999). under under Strasser, LET & in (Haldimann decrease anaerobiosis SA apparent and no 2009), was Johnson, there & C among (Stepien common treatments. salt is NaCl 2003), decrease under Such rates 5). transfer (Figure levels electron linear the the in in decrease changes by shown clearly In is role protective The both of in exposure of decline days content dramatic Chl 12 a the after with mM, 58% 100 stomatal than and both lower 42 concentrations and NaCl 4B) at even (Figure comparison, did weeks; By mM leaves of 250 data). content unpublished below Chl al., concentrations The NaCl mortality. ( significant at without conductance particularly NaCl significantly, mM drop 550 roles. to not earlier protective up role, these concentrations provides protective with NaCl hence the together under and of study, pool this reflection PQ from a over-reduced data As oxidize The can 2007). tomato PTOX al., of that et mutant suggests (Shahbazi the studies, (SM) on Marzano San treatment control in light over-reduction the high pool PQ of high effect a and similar limitation side acceptor PSII strong to a to leads This 1999). Q pronounced hw) fe xouet atsrs,teJse fOI uvswssgicnl nacdfor enhanced significantly F was curves stress. OJIP salt of under J-step the PTOX 2013), stress, of salt al., role to in to et protective increase exposure Laureau the After to for 2005; shown). found evidence al., was new et (OJIP) provide (Streb fluorescence study exposure this UV stress. from 2009) under 2007; high Johnson, Data PTOX al., & and of (Stepien et temperature salinity role Diaz 2012), low generic 2006; al., at a (Quiles, et (Ivanov implying drought plants light and alpine high light and in high temperatures and low temperatures, 2010), high PQH al., as pool, et such Ibanez in plastoquinol stress conditions the abiotic salt Kuntz, of to under & the oxidation exposed PTOX (Carol of the of biosynthesis stromal-side increase in carotenoid the the the implicated in to to been involved attached is has stress 2003) PTOX and Stenmark, salt 2003). 2001) & al., to et (Berthold thellungiella (Lennon protein membrane membrane or thylakoid interfacial 2007) an al., is PTOX plants et in in valve (Shahbazi safety earlier light a reported as high PTOX and been to 4.2 RNA also tomato have PTOX of levels of 2009). PTOX exposure amount &Johnson, of (Stepien increased increase e.g. were The stress, there TPOX. under into stress, flow salt electron under in increased that sink in showed electron abundance also major protein a 8) as (Figure O functions low analysis may under PTOX observed that not rate was evidence transfer further electron provides This SA PSII minimized. primary O is the 21% power in of enhancement presence an the CO observed in we salt O possibility, reduce O under to this 21% used with of be presence Consistent can electrons the stress, at salt severe especially photoinhibited, in that less observed was we DBMIB, using Thirdly, SV, SA SA 2 ne atsrs.Frhroe nln ihti oain hsehneeto lcrntase rate transfer electron of enhancement this notation, this with line in Furthermore, stress. salt under ne teswswl rtce.Cnitn ihteedffrnilcpcte opoetphotosystem protect to capacities differential these with Consistent protected. well was stress under h u hog htrsiainmnmzs ec h htrsiaina ao ikfrreducing for sink major a as photorespiration the hence minimizes, photorespiration through flux the , nti ead iia eut aebe eotdb hhaie l 20) hs uhr proved authors These (2007). al. et Shahbazi by reported been have results similar regard, this In . dt o hw) h nrae ee sa niao famr eue Qpo n more a and pool PQ reduced more a of indicator an is level J increased The shown). not (data ne atsrs,w bevdadces nlna lcrntase ae(E)in (LEF) rate transfer electron linear in decrease a observed we stress, salt under - gs A g piayeeto cetro SI cuuainudrsl tes(admn Strasser, & (Haldimann stress salt under accumulation PSII) of acceptor electron (primary n siiaina topei CO atmospheric at assimilation and ) ETC SA tstrtn CO saturating at u o in not but , 2 2)udrsl tes(iue5,B.Tegn xrsinadWesternblot and expression gene The B). 5A, (Figure stress salt under (2%) gs SA and 2 ne atsrs opoetpooytm rmover-reduction from photosystems protect to stress salt under SV SA 2 SV n auaigCO saturating and SV A hc a ocnrto f2000 of concentration a has which , Esmn ta. nulse data). unpublished al., et (Essemine lnsge omlya oeaesl tesadee survived even and stress salt moderate a at normally grew plants Fgr ) hrfr,uo atsrs,the stress, salt upon Therefore, 8). (Figure a nbet uvv tNC ee ihrta 0 Mfrtwo for mM 100 than higher level NaCl at survive to unable was o5 n 0 MNC,rsetvl Fgr A,concurrent 4A), (Figure respectively NaCl, mM 100 and 50 to SA, SV SA, scmae to compared as u a o hne rcagdltl for little changed or changed not was but 2 3 in 9 h TXlvl aebe on oices nplants in increase to found been have levels PTOX the pce ne tes ..dogt(odn Johnson, & (Golding drought e.g. stress, under species SA 2 ocnrtos()wr anand(seieet (Essemine maintained were (A) concentrations 2 hog TXwtotpsigtruhCytb through passing without PTOX through 2000 , 2 ghost SV Fg7,D;ti spsil eas under because possibly is this D); 7C, (Fig SV μ oprdto compared L L ne ihNC ramn,tePSII the treatment, NaCl high under , ( gh -1 eetv nPO oprdto compared PTOX in defective ) SV Fgr B.Udr20 ml 2000 Under 5B). (Figure μ L L rpe rsial yabout by drastically dropped 2 Je ta. 02.Similar 2002). al., et (Joet SA -1 tete 1 r2 O 2% or 21% either at vntog h salt the though even , SA SV, hw drastically shows SA ssonb the by shown as SV SA SV ncontrast, in , o compared compared dt not (data fChl of · L 6 -1 f. a 2 Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. Data may be preliminary. tlt fgnsfo hshlpyet mrv rpsl oeac Biahe l 02 aa Subudhi, & Karan 2012; al. et (Baisakh the tolerance demonstrated salt have crop studies improve to ealier halophyte Several this 2011). here from Baisakh, mentioning genes & worth of (Subudhi is utility genes It stress-responsive elucidation. salt further mine carbon needs such photosynthetic NaCl chloroplast, the under in from that regulated compound signals differentially particular internal NDH were or some of NDH-CET chain, PTOX, that H phosphorylation transfer and even electron even possible NDH or chloroplast the and metabolism, is in of 2003), It changes status differential unknown. al., redox induced as complexly et NaCl that is Lascano likely mechanism is 2012; It though al., 2003). 2001; al., et H al., et (Terashima et (Lascano 2013), (Lasano signaling components al., status Ca redox et including a 2010), Takahashi mechanisms, showed al., 2006; of also array al., in an (2002) activity by et (PQH2) The al. regulated Arabidopsis. Breyton is plastoquinol Jo¨et expressing from et PTOX transfer 2014); tobacco for expressing electron lines al., cyclic in CEF transgenic of earlier tobacco activity et with in of NDH (Galzerano CEF efficiently number NDH-dependent in lines in competes a decrease decline desaturase) with PTOX dramatic (carotene line both (Cr-PTOX1); CRTI-expressing a in by algae the demonstrated is assessed green (2012) NDH-CEF as from al. and control PTOX PTOX et between the Ahmad relationship to e.g. negative reports, compared this However, times of 8). finding 4 8). and Our to (Figure 6 abundance up (Figs. protein by level and PTOX up-regulated abundance stable was RNA-expression nearly which a levels, to expression concurrent CEF was NDH NDH-dependent assay (236%) of in to stress ( responses here the species salt in used between following contrast was stress stark which CEF a salt This signal, found 6). to (PIR) we (Figure PTOX method, rise in decreased Fo this and that Using rate Shikanai post-illumination show 1998). transfer the 2006; al., we electron from et al., cyclic here (Burrows data of contrast, et by rate shown In (Brayton the clearly 2015). PTOX, increases is al., to usually electrons et channeling rate Strend of transfer 2013; capacity al., electron et cyclic Takahashi the 2007; conditions, transfer stress electron Under cyclic levels; NDH-mediated light and and PTOX temperature 4.3 different under rate al., assimilation (Jo¨et O et 2005). carbon Arabidopsis 21% for from species under electrons PTOX oxygen of especially of over-expressing sensitivity reactive tobacco consumption enhanced in of the the shown However, rate species when not mountain 2009). photoprotection high was to al., In light sufficient et led 2002). high (Heyno PTOX a detoxified to of authors provide effectively growth the over-expression be plant which only however, the can or can tobacco, PTOX in light, by PTOX high In PTOX using generated under of studied the vulnerability 2006). role been increased that a al., but also for suggest light et has low evidence (Rosso increased Arabidopsis under provide status photoprotection in to not redox increased PTOX led did in however, PQ of it resulted which of ortholog light, lines; it The regulation high over-expression 2012), to and tobacco. al., mutant When resistance for et both increased light conclusive. (Ahmad inducing not high tobacco of still to are into vulnerability instead experiments transferred furthermore, transgenic transgenic was retardation; through from growth earlier reinhardii far studied Chlamydomonas been so from has obtained PTOX PTOX data of function the linear protective leaves. However, the photosynthetic in that approaches. the ROS here to of mentioning of worth contributed maintenance accumulation is may by less It 8) shown and (Figure content, as a salt function, chlorophyll under and transfer, activity structure electron PTOX chloroplast higher of having in protection that photosystem the radical suggest for to anion data structure salt chloroplast superoxide these of the the Altogether, damage in 1971), severe swelling (Kearns, The a oxygen by 1997). singlet (Fridovich, reactive peroxide highly hydrogen include and may here detected ROS treat to used concentration SA SA eosre eln nteNHdpnetCF(iue6 oehrwt nices fPTOX of increase an with together 6) (Figure CEF NDH-dependent the in decline a observed we , a enue samdlhlpyegasseist td dpaint lnst atsrs n to and stress salt to plants to adaptation study to species grass halophyte model a as used been has 2 O 2 2 n ihC high and ih ieetal euaePO n D-E.Mcaim o TXand PTOX how Mechanisms NDH-CET. and PTOX regulate differentially might , SV auclsglacialis Ranunculus a 0m,wieta sdt treat to used that while mM, 50 was i ugsigamjrrl fPO npoortcin(te tal., et (Streb photoprotection in PTOX of role major a suggesting , SA SV and 2 fe xouet at(seiee l,upbihddata). unpublished al., et (Essemine salt to exposure after O SV 2 10 Srn ta. 05,mtblt ees(iigtnet (Livingston levels metabolite 2015), al., et (Strand h aeo h iereeto rnfrfrecesthe exceeds far transfer electron linear the of rate the , .In ). SV h togsiuaino NDH-dependent of stimulation strong the , SA a 5 M(iue9 0.The 10). 9, (Figure mM 250 was SA SV hc a great a has which , sas reflected also is Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. Data may be preliminary. ao,P,&Knz .(01.Apatdtria xds oe olgt mlctosfrcaroteno- for implications light: to comes oxidase terminal plastid A functional (2001). chlororespiration. 1385(00)01811-2 a and M. of ndh biosynthesis Kuntz, Identification of plastid id (1998). & disrupted modulation J. P., containing P. Redox mutants Nixon, Carol, tobacco & (2006). of P. analysis Maliga, G. through Z., chloroplasts genes. Finazzi, Svab, in C A., & complex L. in respiratory P., Sazanov, A., I Joliot, P. Burrows, N., photosystem G. around Johnson, flow ps://doi.org/10.1021/bi061439s B., electron Nandha, cyclic based C., genome Maggio, a & species: Breyton, J., extremophile H. in Bohnert, G., stress Yun, salt G., counteract Inan, that M., view. Dassanayake, mechanisms D., for Oh, Biotechnology F., (2013). Orsini, A. C., H. Park, A., R. and Bressan, photosynthesis on impact Salt (2007). A. Smaoui, 007-0094-z & of C., Abdelly, ultrastructure W., leaf Chaibi, & W., H Djebali, C., Z., vacuolar Vanier, Barhoumi, a D., expressing grass Galbraith, plants halophyte J., rice the https://doi.org/10.1111/j.1467-7652.2012.00678.x Janda, of 453-464. from P., tolerance gene stress Subudhi, (SaVHAc1) salt K., c1 Enhanced Rajasekaran, (2012). V., A. M. Pereira, Ramana-Rao, N., plants. Baisakh, Arabidopsis variegated in The sectors (2001). green S. 10.1104/pp.127.1.67 and doi: Rodermel, white 67-77. & of morphogenesis D., the Wu, and H., differentiation Bae, R., M. Aluru, in proteins membrane foreign oxidase. of terminal production plastid the algal nal.pone.0041722 Investigating an (2012). of J. expression P. chloroplasts: Nixon, tobacco & ornamental F.. Michoux, the N., of Ahmad, mechanisms biochemical and Physiological lia P., (2015). Diaz-Vivancos, A. Hern´andez, R., J. J. Acosta-Motos, declare. to interest References of conflicts no have primers. authors and The sequences genes other some and video Disclosures one Tables, two includes document This INFORMATION SUPPORTING dismutase; superoxide SOD, P oxidase; PQH terminal plastoquinone; PQ, quenching; non-photochemical NPQ, dehydrogenase; ( g gallate close); RCs (PSII fluorescence maximum ’daioezdn;DMB ,-irm--ehl6iorplp ezqioe T,eeto transport electron induction; ETC, fluorescence benzoquinone; FI, stress. chain; 2,5-dibromo-3-methyl-6-isopropyl-p- salt DBMIB, under diaminobenzidine; photosystems photosystem 3’ protect protect to to strategy NaCl : new under Abbreviations develop respond help NDH-CET can and functioning PTOX leaf how of and elucidation Therefore, b). 2012a, .pat o oigwt alsrs n recovery. and stress NaCl with coping for plants L. ln itcnlg Reports Biotechnology Plant MOJournal, EMBO n P,345tiyrx-ezi cdnpoy se) B,ntolettaoim D,NAD(P)H NDH, tetrazolium; nitroblue NBT, ester); acid-n-propyl 3,4,5-trihydroxy-benzoic -PG, A htsnhtcrt;CT aaae E,cci lcrnflw h,clrpyl A,3, DAB, chlorophyll; Chl, flow; electron cyclic CEF, catalase; CAT, rate; photosynthetic , 700 euou littoralis Aeluropus htsse ecincne;RS ecieoye pce;S,strtn pulse; saturating SP, species; oxygen reactive ROS, center; reaction I photosystem , 74,8886 o:10.1093/emboj/17.4.868 doi: 868-876. 17(4), SV gs , eai viridis Setaria ,2-7 o:10.1007/s11816-012-0249-9 doi: 27-37. 7, , tmtlcnutne F conductance; stomatal , rnsi ln Science Plant in Trends . ETC ora fPatResearch Plant of Journal lae,S,Fr´ne-acı,N,SnhzBac,M . & J., M. Sanchez-Blanco, Fern´andez-Garc´ıa, N., S., Alvarez, ` odcac fteeeto rnfrchain; transfer electron the of conductance , priaalterniflora Spartina ; SA 11 3 Planta , plants. priaalterniflora Spartina o 4,8986 doi:10.1007/s00425-015-2315-3. 829-846. 242, , iiu ursec PI C pn;F open); RCs (PSII fluorescence minimum , Biochemistry ,3-6 https://doi.org/10.1016/S1360- 31-36. 6, , LSOne PLoS Loisel. 2,5957 o:10.1007/s10265- Doi: 529-537. 120, . immutans ln itcog Journal Biotecholgy Plant . 2 lsounl TX plastid PTOX, plastoquinol; , ,e12.di 10.1371/jour- doi: e41722. 7, , 54) 36-37.htt- 13465-13475. 45(45), , uainaet plastid affects mutation ln Physiology Plant + uei myrtifo- Eugenia n APs subunit -ATPase -PG, n -propyl 127, , 10, , m , Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. 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F). 550 2 and and to D value to up (B, numerical normalized for Spartina and were a salt E) parameters giving and test by C JIP standardized (A, of was data reference and 3 All the (A-C) FIGURE mM at days. 100 variable 12 and each (1). for 50 unit and NaCl 0, the NaCl (D-F) with of mM mM treated 0 550 D-F) reference and column, the (right 400 Spartina 250, and 100, A-C) 0, column, (left Setaria for respectively. 2 modulation, FIGURE amplitude pulse and analyzer gas infrared CO controlling for O PAM, O the appropriate of system the emitter-detector 2000 the or on (390 holding chamber a through PAM-100, 1 FIGURE subunit PSI-G the lacking legends I Photosystem Figures photodamage. (2005). to V. sensitive is H. and Scheller, doi:10.1016/j.bbabio.2005.02.003 plastocyanin & 154-163. for D., affinity higher Leister, a E., a has P. in II Jensen, Photosystem A., from Zygadlo, superoxide of Production U. (2014). Yoon, H. M., C. 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Wollman, S., Clowez, H., Takahashi, 2 ocnrtosi h hme uigmaueet.Seas ie o etn.IG n A mean PAM and IRGA setting. for video also See measurements. during chamber the in concentrations rz sativa Oryza iciiae ipyiaActa Biophysica et Biochimica ln Cell Plant immutans h ln Journal, Plant The ceai ersnainsoigteeprmna etn fL-o 40tgte ihDual- with together 6400 Li-cor of setting experimental the showing representation Schematic hne nla Na leaf in Changes sie lt fslce aaeesdrvdfo h hoohl ursec JPcurves OJIP fluorescence chlorophyll the from derived parameters selected of plot’ ‘spider A μ L L . uatlcigPsbS. lacking mutant L.) 2 -1 n sn noye oreeupe iha xmtrt dutoye u from flux oxygen adjust to oxymeter an with equipped source oxygen an using and ) ore( r2% otecabradacrigyb bet oio h CO the monitor to able be accordingly and chamber the to 21%) or (2 source aigto uatof mutant variegation 11,4-5 o:10.1105/tpc.11.1.43 doi: 43-55. 11(1), , + 4 7-0.di 10.1146/annurev.arplant.54.031902.134831. doi: 575-603. 54, , dg . osyia . rmet . G K., Trompelt, I., Tolstygina, M., udig, ¨ ,1115 o:10.1046/j.1365-313x.1994.6020161.x doi: 161-175. 6, AB,K (A-B), 87 1024.doi:10.1016/j.bbabio.2012.08.006. 2140-2148. 1817, , rnsi ln Science Plant in Trends M ln Biology Plant BMC + Arabidopsis CD otnsadK and contents (C-D) aueCommunications Nature rceig fteNtoa cdm fSciences of Academy National the of Proceedings 16 spatdatnmu n mardi carotenoid in impaired and autonomous plastid is () 67.https://doi.org/10.1016/S1360- 66-71. 6(2), , 4 4.http://dx.doi.org/10.1186/s12870- 242. 14, , + /Na iciiae ipyiaActa Biophysica et Biochimica ,15.di 10.1038/ncomms2954 doi: 1954. 4, , bli,P,Ffzn . ul,J., Kudla, C., Fufezan, P., ¨ abelein, + nulRve fPatBiology Plant of Review Annual ai EF vrtm nSetaria in time over (E-F) ratio IMMUTANS + ± SE. rnpr nhge plants. higher in transport aigto locus variegation 109(43), , 1708, , 2 2 level and , Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. Data may be preliminary. nrae AadC rsl rae BadD o eai t5 MadSatn t20m uig12 during mM 250 at Spartina and mM (H peroxide 50 hydrogen at of interaction Setaria the for reflect (500 D) spots light and Brown under DAB (B (diaminobenzidine). with DAB treated mM salt 5 or with days C) radical days and superoxide 12 (A during of mM untreated interaction 250 the at 10 Spartina reveals and FIGURE staining mM 50 Dark-blue at tetrazolium). Setaria for (nitroblue D) NBT ( and mM (B treated 6 salt with or around C) and of basis. (A mean untreated protein the equal 9 an represent on points FIGURE loaded Data blot, PTOX. original The of an methods). analysis from SDS-PAGE and leaves for bands q-PCR western materials nitrogen (B), the (see for liquid level Invitrogen in for replicates Kit expression stored five used Mini immediately gene was RNA and For PureLink cDNA collected using were synthesized purification software. days Western- and 12 technology isolation for membrane. TanonImage plants RNA nitrocellulose by salt-treated 29 to and quantified of control transfer was separation from electrophoretic SDS-PAGE, size and after band samples, immune-detection blot membrane for (for thylakoid 50 treatment and the salt 0 from initiating to after subjected Spartina days 6 and 12 Setaria least (for of at leaves NaCl of mM in 250 means (B) analysis the q-PCR exhibits by point assessed data Each 8 oxygen. FIGURE bars) (black 2% mM and 5 bars) replicates with (gray or 21% bars) of (white presence water with infiltrated vacuum 390 were of presence the in ihrStra( n )o pria( n )dtce rmpat ujce o n 0(for 50 and 0 to: subjected plants from detached D) (for and NaCl (B mM Spartina 250 or and C) and (A Setaria respectively, either Spartina, and 7 Setaria FIGURE for NaCl mM leaves 250 different (325 or light 50 actinic F to with post-illumination subjected The replicates days. or 6 12 (ctrl) least during at medium of salt-free means either the represents point 6 data Each FIGURE oxygen. symbols) (closed 2% or symbols) oun ,D n )edrdNC tinls:10m o eai n 5 MfrSatn.Control Spartina. after for days mM 12 250 performed, and were measurements Setaria CO The for saturating medium. mM under NaCl-free 100 treatment, a (triangles): salt in NaCl initiating maintained endured were H) (circles) and plants F D, B, at column, treatment ETR salt and initiating (E-F), replicates after 6 days 5 least 16 at FIGURE out of means carried the were represents Measurements bar concentration. (D). data NaCl chlorophyll replicates mM 10 determine 400 least to 25 and Fig. at treatment 0 in of or salt described mean (C) (NPQ as initiating the NPQ levels represents after of NaCl bar days components different data 12 to each chlorophyll exposed collected For were were Spartina Leaves month-old 2. two and Setaria month-old 4 FIGURE . O ° ntepeec f390 of presence the in C - 2 ihNT(500 NBT with ) ± SE. ffc fsl ramn nPO rti xrsin()adtePO eeepeso level expression gene PTOX the and (A) expression protein PTOX on treatment salt of Effect h ffc fsl ntela oa hoohl otn nStra()adSatn B.One (B). Spartina and (A) Setaria in content chlorophyll total leaf the on salt of effect The ffcsof Effects D-eedn E aha sesda h otilmnto F post-illumination the as assessed pathway CEF NDH-dependent xgndpnec feeto rnpr:ETR transport: electron of dependence Oxygen itceia tiigo eai CD n pria(-)lae bandfo control from obtained leaves (A-B) Spartina and (C-D) Setaria of staining Histochemical itceia tiigo eai CD n pria(-)lae bandfo control from obtained leaves (A-B) Spartina and (C-D) Setaria of staining Histochemical ± I SE. GH,maue nlae fStra(etclm,A ,EadG n pria(right Spartina and G) and E C, A, column, (left Setaria of leaves in measured (G-H), SA .Frpoenepeso A,lae rmcnrladsl-rae lnswr collected were plants salt-treated and control from leaves (A), expression protein For ). μ μ μ SA o m mol L L n o m mol P n BI nPI htceia efficiency photochemical PSII on DBMIB and -PG f .Maueet eecridot1 asatriiitn atteteta 25 at treatment salt initiating after days 12 out carried were Measurements ). -1 μ μ n Ps nlae fStra()adSatn D xoe ot n 100 and 0 to to exposed (D) Spartina and (C) Setaria of leaves in NPQs) and o m mol L L CO -2 -2 s s -1 -1 2 -1 evswr luiae ih800 with illuminated were Leaves . .Ec aabrrpeet h en fa es 0rpiae ae on taken replicates 10 least at of means the represents bar data Each ). nlae olwn atsrs treatment. stress salt following leaves in ) CO -2 s -1 ± 2 o evswr luiae ih800 with illuminated were Leaves . nlae olwn atsrs treatment. stress salt following leaves in ) Ead6rpiae o R-C.Isr fpnlAsostypical shows A panel of Insert qRT-PCR. for replicates 6 and SE sewsrcre ntedr fe wthn ff5mnillumination min 5 off switching after dark the in recorded was rsie 2 (2000 17 ± μ SE. L L -1 ,a 25 at ), II n P AB r50 or (A-B) -PG AB,P (A-B), ° n ntepeec f2%(open 21% of presence the in and C μ o m mol 700 μ o m mol ± -2 xdto ai (C-D), ratio oxidation Φ E at n slow-relaxing and Fast- SE. s -1 PSII o μ e cii ih.Leaves light. actinic red -2 BI CD nthe in (C-D) DBMIB M iei lnsgonon grown plants in rise s esrdi evsof leaves in measured -1 cii ih.Each light. actinic SV rt and 0 to or ) SV μ protein g r0 or ; ) g ± 2 ETC O SE. ° 2 C ) Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. Data may be preliminary. 18 Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. Data may be preliminary. 19 Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. Data may be preliminary. 20 Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. Data may be preliminary. 21 Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. Data may be preliminary. 22 Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. Data may be preliminary. 23 Posted on Authorea 12 Mar 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158398128.82351300 — This a preprint and has not been peer reviewed. Data may be preliminary. 24