Plan Physiology and Biochemis y
Dis inc non-pho ochemical quenching cha ac e is ics unde cons an and luc ua ing
ligh condi ions: compa ison be ween local and impo ed obacco a ie ies
--Manusc ip D a --
Manusc ip Numbe :
A icle Type: Resea ch Pape
Sec ion/Ca ego y: Pho osyn hesis; plan -mic obe in e ac ions (bio ic s ess + bene icial)
Keywo ds: non-pho ochemical quenching; local and impo ed a ie ies; luc ua ing ligh ;
xan hophyll cycle; obacco
Co esponding Au ho : Yingchao Lin
Guizhou Tobacco Science Resea ch Ins i u e
Guiyang, CHINA
Fi s Au ho : Hui Lyu
O de o Au ho s: Hui Lyu
Zhaoqi Tang
Weijie Xu
Dejun Kong
Zhihong Wang
Zhixiao Yang
Jishun Zhang
Hongqi Wu
Zili Wang
Dusan Laza
Yingchao Lin
Abs ac : To explo e he a ia ion in adap abili y o luc ua ing ligh en i onmen s and op imize
hei pho osyn hesis and g ow h, he non-pho ochemical quenching (NPQ)
cha ac e is ics o a local obacco a ie y Xiao eng 8 and a o eign in oduced a ie y
K326, unde bo h cons an and luc ua ing ligh condi ions we e examined. Unde high
ligh in ensi ies, Xiao eng 8 exhibi ed a highe NPQ alue compa ed o K326, indica ing
an enhanced capaci y o dissipa ing excess ligh ene gy as he mal ene gy and
mi iga ing he po en ial isk o pho odamage. The induc ion changes o NPQ and
pho osys em II e iciency (ΦPSII) om da k o low and high ligh condi ions we e
analyzed, e ealing dis inc adap abili y o high ligh en i onmen s and associa ed
pho op o ec ion mechanisms in Xiao eng 8. While Xiao eng 8 induced he egula o y
non-pho ochemical quenching in high ligh , K326 ealized he p o ec ion by he
cons i u i e non-pho ochemical quenching, which p ese ed also o low ligh condi ions
and seem o be he eason o a lowe g ow h o K326 han o Xiao eng 8 in na u al
ligh condi ion, i.e., unde luc ua ing ligh . Addi ionally, he ole o xan hophyll cycle
pigmen s in egula ing NPQ and ene gy dissipa ion unde high ligh in ensi ies was
examined using mu an s de ec i e in iolaxan hin de-epoxidase (VDE) and zeaxan hin
epoxidase (ZEP). The esul s showed ha de ec s in hese enzymes a ec ed he NPQ
esponse and educed he g ow h a e o he mu an s unde luc ua ing ligh condi ions,
sugges ing a c ucial ole o he xan hophyll cycle in plan g ow h and adap a ion o
a iable ligh en i onmen s. Collec i ely, hese indings emphasize he impo ance o
explo ing gene ic esou ces imp o ing plan NPQ unde luc ua ing ligh condi ions
om bo h domes ic and impo ed a ie ies, he eby p o iding p omising a ge s o
selec ing and b eeding new c op a ie ies wi h imp o ed yield, quali y, and esis ance,
ul ima ely enhancing b eeding e iciency and c op adap abili y.
Powe ed by Edi o ial Manage ® and P oduXion Manage ® om A ies Sys ems Co po a ion
1/25
Dis inc non-pho ochemical quenching cha ac e is ics unde cons an and luc ua ing ligh
condi ions: compa ison be ween local and impo ed obacco a ie ies
Hui Lyu1,†, Zhaoqi Tang2,†, Weijie Xu2, Dejun Kong3, Zhihong Wang3, Zhixiao Yang3, Jishun Zhang3,
Hongqi Wu4, Zili Wang3, Dusan Laza 5, Yingchao Lin3,*
1 School o Biological Science and Ag icul u e, Qiannan No mal Uni e si y o Na ionali ies, Duyun,
China
3 Guizhou Academy o Tobacco Science, Guiyang, China
2 Shanghai Tobacco G oup Co., L d, Shanghai, China
4 College o Tobacco Science, Guizhou Uni e si y,Guiyang, China
5 Depa men o Biophysics, Facul y o Science, Palacky Uni e si y, Olomouc, Czech Republic
* Co espondence: [email protected]
† These au ho s con ibu ed equally o his wo k.
Two geno ype NPQ by YL.docx Click he e o iew linked Re e ences
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Abs ac : To explo e he a ia ion in adap abili y o luc ua ing ligh en i onmen s and op imize hei
pho osyn hesis and g ow h, he non-pho ochemical quenching (NPQ) cha ac e is ics o a local obacco
a ie y Xiao eng 8 and a o eign in oduced a ie y K326, unde bo h cons an and luc ua ing ligh
condi ions we e examined. Unde high ligh in ensi ies, Xiao eng 8 exhibi ed a highe NPQ alue
compa ed o K326, indica ing an enhanced capaci y o dissipa ing excess ligh ene gy as he mal ene gy
and mi iga ing he po en ial isk o pho odamage. The induc ion changes o NPQ and pho osys em II
e iciency (ΦPSII) om da k o low and high ligh condi ions we e analyzed, e ealing dis inc
adap abili y o high ligh en i onmen s and associa ed pho op o ec ion mechanisms in Xiao eng 8. While
Xiao eng 8 induced he egula o y non-pho ochemical quenching in high ligh , K326 ealized he
p o ec ion by he cons i u i e non-pho ochemical quenching, which p ese ed also o low ligh
condi ions and seem o be he eason o a lowe g ow h o K326 han o Xiao eng 8 in na u al ligh
condi ion, i.e., unde luc ua ing ligh . Addi ionally, he ole o xan hophyll cycle pigmen s in egula ing
NPQ and ene gy dissipa ion unde high ligh in ensi ies was examined using mu an s de ec i e in
iolaxan hin de-epoxidase (VDE) and zeaxan hin epoxidase (ZEP). The esul s showed ha de ec s in
hese enzymes a ec ed he NPQ esponse and educed he g ow h a e o he mu an s unde luc ua ing
ligh condi ions, sugges ing a c ucial ole o he xan hophyll cycle in plan g ow h and adap a ion o
a iable ligh en i onmen s. Collec i ely, hese indings emphasize he impo ance o explo ing gene ic
esou ces imp o ing plan NPQ unde luc ua ing ligh condi ions om bo h domes ic and impo ed
a ie ies, he eby p o iding p omising a ge s o selec ing and b eeding new c op a ie ies wi h
imp o ed yield, quali y, and esis ance, ul ima ely enhancing b eeding e iciency and c op adap abili y.
Key wo ds: non-pho ochemical quenching; local and impo ed a ie ies; luc ua ing ligh ; xan hophyll
cycle; obacco
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In oduc ion
In na u al en i onmen s, plan s o en ace luc ua ions in ligh in ensi y due o a ious ac o s such as
cloud co e , lea mo emen in he wind, plan shading and changes in sola angle (Long e al., 2006;
Taylo and Long, 2017). This luc ua ion in ligh in ensi y can lead o apid changes in he in ensi y o
ligh ecei ed by plan lea es, ha e a signi ican impac on he pho osyn hesis o plan s. On he one hand,
luc ua ions in ligh in ensi y can a ec he a e and e iciency o pho osyn hesis in plan s, he eby
a ec ing hei g ow h, de elopmen , and yield (Sla e y e al., 2018). On he o he hand, wi h he apid
inc ease in ligh in ensi y, when plan s abso b mo e ligh ene gy han hey need o pho osyn hesis, he
excess ligh ene gy may cause damage o plan cells (Shi e al., 2022; Sha iq e al., 2021).
To cope wi h such challenges, plan s ha e e ol ed a ious pho op o ec i e mechanisms, among
which he ene gy dependen non-pho ochemical quenching (qE) plays a c ucial ole (Bielczynski e al.,
2017; Shi e al., 2022; Lu e al., 2022; Niu e al., 2023). The qE is a apidly inducible p ocess ha enables
plan s o dissipa e excess abso bed ligh ene gy as hea , he eby p o ec ing hei pho osyn he ic appa a us
om pho odamage (Nicol e al., 2019). The qE equi es low pH o hylakoid lumen o p o ona e PSII
subuni S (PsbS) (B ooks e al., 2014; Li e al., 2000) and o ac i a e iolaxan hin-deepoxidase (VDE),
which con e s iolaxan hin (V) ia an he axan hin (A) o zeaxan hin (Z) (Demmig-Adams 1990;
Yamamo o e al., 1962).
Due o di e en gene ic cha ac e is ics and pho osyn he ic mechanisms, he e a e di e ences in he
qE mechanisms o plan a ie ies om di e en egions and geno ypes (Tanaka e al., 2019). Fo example,
o e exp ession o h ee ansgenes (A VDE, A PsbS, and A ZEP) in soybean and obacco, which a e
equi ed o enhance se ing and decay o qE unde high ligh o apidly luc ua ing condi ions as widely
desc ibed p e iously, accele a ed he elaxa ion o NPQ du ing sun- o-shade ansi ions, esul ing in
inc eased pho osyn he ic e iciency and c op p oduc i i y unde luc ua ing ligh (K omdijk e al., 2016;
De Souza e al., 2022). A he same ime, i was ound ha some ice and co n a ie ies ha e highe NPQ
abili y, which can be e dissipa e excess ligh ene gy, p o ec he pho osyn he ic sys em om ligh
damage, main ain high pho osyn he ic a e and yield, while o he a ie ies canno e ec i ely cope wi h
high ligh induced pho odamage, esul ing in a dec ease in pho osyn he ic a e and yield (Li e al., 2020;
Wang e al., 2017; Tsai e al., 2019). The e a e di e ences in he NPQ abili y o plan a ie ies wi h
di e en o igins o high ligh , which a e mainly due o he di e en gene ic backg ounds, and
e olu iona y his o ies o hei espec i e g ow h en i onmen s. Unde s anding hese di e ences helps us
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o be e unde s and he pho osyn hesis mechanism and pho op o ec ion s a egies o plan s, p o iding
scien i ic basis and echnical suppo o ag icul u al p oduc ion. While NPQ has been ex ensi ely
s udied unde s able ligh condi ions, i is impo an o no e ha plan s o en ace luc ua ions in ligh
in ensi y in na u e, as desc ibed ea lie . The e o e, unde s anding he NPQ esponse o plan s unde
luc ua ing ligh condi ions is o g ea signi icance. By s udying he esponse o NPQ o luc ua ing ligh ,
we can be e unde s and how plan s adap o hese en i onmen al changes, and how o imp o e plan
s ess esis ance, pho osyn he ic e iciency, and yield h ough gene ic enginee ing and o he means,
p o iding new solu ions o ensu ing global ood secu i y.
Tobacco is well-known as a smoking ma e ial bu i has always been conside ed as a medicinal plan
and used as a adi ional medicine o common illnesses (Cha l on 2004, Be lowi z e al. 2020). In p esen
knowledge, nico ine con ained in obacco is an agonis o ace ylcholine, he la e being na u al chemical
in ol ed in ligand-ga ed nico inic ace ylcholine ecep o s, i.e. ligand-ga ed ion channels. These ion
channels a e indispensable o p ope ansmission o elec ic signals in synapses be ween neu ons and
neu on and muscle cells (Hogg e al. 2003) Tobacco plan s can be also used as a na u al li ing labo a o y
o p oduce human he apeu ic eagen s (Sanchez-Ramos 2020). These ac s indica e ha plan ing obacco
migh be use ul bu i is impo an o know, which a ie y o obacco bes su i es in he en i onmen .
Xiao eng 8 is a common obacco a ie y in Guizhou, China. I is cha ac e ized by a mo e obus g ow h
a e in he ield and a highe p oduc i i y o biomass. On he o he hand, K326 obacco a ie y was
expo ed o China. This a ie y is esis an o he common oo -kno nema odes and i is known o high
quali y and cu abili y (see h ps://c ossc eekseed.com/usa- a ie ies/usa- lue-cu ed- obacco-seed/k-326/).
In his wo k, we s udied i he seemingly supe io K326 a ian is be e in g own in he ield, i.e.,
unde luc ua ing ligh condi ions. Namely, we in es iga e he NPQ cha ac e is ics o wo obacco
cul i a s, Xiao eng 8 and K326, unde bo h cons an and luc ua ing ligh condi ions. By compa ing hei
NPQ esponses, we aimed o gain insigh s in o how plan s adap o luc ua ing ligh en i onmen s and
op imize hei pho osyn hesis and g ow h. Fu he mo e, we also examined he ole o xan hophyll cycle
pigmen s in egula ing NPQ and ene gy dissipa ion unde high ligh in ensi ies. Ou indings p o ided
aluable insigh s in o he pho op o ec i e s a egies o plan s and ha e implica ions o c op b eeding
and gene ic enginee ing o imp o e pho osyn he ic e iciency and yield unde luc ua ing ligh condi ions.
Ma e ials and me hods
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Plan ma e ials and g ow h condi ions
Fo he ield expe imen , he obacco cul i a s Xiao eng 8, a local land ace, and K326 ha in oduced
om USA, we e selec ed as s udy ma e ials on he basis o plan size and g ow h a e acco ding o
p e ious obse a ions. The ial was conduc ed in Qingzhen (la . 26°31′27″N, long. 107°21′16″E) in
2022, a an al i ude o 1280 m, in Guizhou, China. Ten-day a e age empe a u e and 10-day sunshine
hou eco ds om Ma ch o Oc obe a e p esen ed in Fig. 1. No e: wha is 10-day a e age empe a u e
and en-day sunshine hou ? Speci y i di e en ly so ha i is clea wha you mean. The soil p ope ies
(0–20 cm) a he s udy si e a e p esen ed in Lin e al. (2020). Seeds we e sown in an open g eenhouse on
25 Feb. 2022 on ansplan ing disks as desc ibed p e iously in de ail (Lin e al., 2017). A se ies o
ag icul u al ope a ions, including u ning up he soil, idging, plas ic mulching, and plan ing hole
p epa a ion, we e ca ied ou as desc ibed p e iously in de ail (Lin e al., 2015, 2017). Seedlings we e
ansplan ed in o he es plo s (15×7 m) wi h h ee eplica es o each geno ype on 26 Ap . 2023. Fo
de ailed obse a ions and measu emen s, i een plan s we e andomly selec ed om bo h cul i a s.
The de (LOC104218706) and zep (LOC104218557) knockou mu an we e gene a ed om Bio un
Biosciences (Wuhan, China) by CRISPR–Cas9, using a single-guide RNA. To in es iga e he e ec s o
xan hophyll cycle on seedlings pigmen s, NPQ cha ac e is ic and g ow h unde luc ua ing ligh
condi ions, con i med de and zep knockou mu an seeds we e sown as desc ibed p e iously in de ail
(Lin e al., 2017). Seedlings ha we e 45-day old we e ansplan ed o plas ic po s (diame e 7 cm; heigh
7 cm) illed wi h o ganic soil and main ained in a g ow h chambe wi h a 16/8 ligh /da k cycle. A cons an
ligh in ensi y o 1000 µmol pho ons m-2 s-1 was se o pigmen s and NPQ cha ac e is ic in es iga ion
while he ligh in ensi y o g ow h pheno ype expe imen was se o luc ua e e e y 3 min, andomly
among 70, 400, 700, 900 µmol m−2 s−1 wi h an o e all ac ional con ibu ion o 0.1, 0.3, 0.4, 0.2,
espec i ely, which ensu ed he a e age ligh in ensi y was close o 600 µmol pho ons m-2 s-1.
Chlo ophyll a luo escence measu emen s
Chlo ophyll a luo escence (ChlF) was measu ed using a po able gas exchange sys em LI-6400XT (Li-
Co Inc., Lincoln, NE, USA). The kine ics we e i s eco ded on a ached lea es o 30 min da k-adap ed
plan s using ac inic ed ligh 70 µmol pho ons m-2 s-1 (low ligh ) o 1000 µmol pho ons m-2 s-1 (high ligh )
o 6 min. Fo expe imen s in luc ua ing ligh , a ached lea es o 30-min da k-adap ed plan s we e
illumina ed o ou cycles o 3 min low ligh (70 μmol pho ons m−2 s−1) and 3 min high ligh
(1000 μmol pho ons m−2 s−1), NPQ induc ion and elaxa ion we e moni o ed a 20 s in e als.
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The ollowing pa ame e s we e de e mined om measu ed ChlF alues (see Lazá 2015 o a e iew):
e ec i e quan um yield o PSII pho ochemis y in ligh -adap ed s a e (ΦPSII) calcula ed as (Fm′–F)/Fm′,
ela i e PSII elec on anspo a e (ETR) as ΦPSII×PAR×0.84×0.5, and non-pho ochemical quenching
pa ame e (NPQ) as (Fm–Fm′)/Fm′, whe e Fm is maximum ChlF in da k-adap ed s a e, Fm′ is maximum
ChlF in ligh -adap ed s a e, and F is ChlF in he ac inic ligh .
Assays o Plan g ow h
Lea a ea was measu ed using he esh lea by A ea me e (AM 300, ADC Bio-scien i ic L d., UK) and
eplica ed o h ee imes wi h 5 seedlings pe eplica e. Numbe o lea es was su eyed a di e en ime
o lea numbe de e mina ion. Plan heigh was measu ed by he dis ance om he i s lea base om
bo om o he g owing poin s o shoo s ( he apical me is ems). The d y weigh (DW) o he lea es was
de e mined using he mass me hod a e d ying he plan s a 105°C o 48 h.
Lea pigmen analysis
Pigmen s we e analysed by e e se-phase-pe o mance liquid ch oma og aphy. Fo pigmen s ex ac ion,
lea discs we e ozen in liquid ni ogen and suspended in KOH me hanol solu ion (20%), keep a 55 °C
o 30 minu es. Ex ac ion solu ion (ace one: e hyl ace a e=1:2, / ) was added a e cooling and
ul asonic ex ac ion o 40 minu es. A e cen i uge o 15 (4 ℃, 8000 g), The supe na an was
e apo a ed o d yness. A e dilu ed wi h ace one, ex ac ed pigmen s we e il e ed h ough a memb ane
il e (po e size 0.2 μm), and used o high-pe o mance liquid ch oma og aphy analysis. The con en s
o Lea pigmen s we e measu ed simul aneously using a Welch Ul ima e C30 (5 μm, 4.6×250 mm)
(Welch, Shanghai, China) wi h de ec ion wa eleng h and column low a e we e 450nm and 1.0ml/min,
espec i ely. The xan hophyll cycle de-epoxida ion s a e (DEPS) has been shown o ha e a be e linea
ela ionship wi h plan hea dissipa ion capaci y, and i was calcula ed as DEPS=(Z+0.5A0/(V+Z+A)
(Johnson e al., 1993).
The da a analysis
Da a analysis so wa e SPSS 17.0 was used o one-way analysis o a iance (ANOVA), and he
signi ican di e ences be ween geno ype we e indica ed wi h di e en lowe case le e s (P≤0.05).
S a is ical da a we e exp essed as a e age alue ± s anda d e o (SE).
Resul s
Seedlings o Xiao eng 8 g ew as e in he ield expe imen
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A e being ansplan ed o 20 d, signi ican pheno ypic di e ences we e obse ed be ween seedlings
o Xiao eng 8 and K326 (Fig. 2A). In e ms o plan heigh , K326 exhibi s a signi ican ly lowe g ow h
han Xiao eng 8, which indica es a mo e igo ous e ical g ow h pa e n in Xiao eng 8 compa ed o
K326 (Fig. 2D). O he g ow h pa ame e s such as lea numbe , maximum lea a ea, and esh weigh o
Xiao eng 8 we e signi ican ly highe han hose ones o K326 (Fig. 2B, C, E and F). O e all, Xiao eng
8 shows a mo e obus g ow h pa e n compa ed o K326.
A s onge abili y o dissipa e excess ligh ene gy in Xiao eng 8 unde high ligh in ensi y
To in es iga e he pho osyn he ic acclima ion o he wo obacco a ie ies o di e en ligh condi ions,
induc ion o NPQ pa ame e and e ec i e quan um yield o PSII pho ochemsi y, ΦPSII, du ing he da k
o low and high cons an ligh condi ions we e analyzed. Du ing he i s 40 seconds on a shi om da k
o low ligh condi ions (70 μmol pho ons m-2 s-1), alue o NPQ pa ame e inc eased and hen g adually
dec eased in K326 seedlings, while he NPQ pa ame e g adually dec eased in Xiao eng 8 seedlings (Fig.
3A). On he o he hand, a apid inc ease in NPQ we e obse ed in bo h K326 and Xiao eng 8 seedlings
on shi om da k o high ligh condi ion (1000 μmol pho ons m-2 s-1), while he s eady s a e alue o
NPQ in Xiao eng 8 was highe han hose o K326 (Fig. 3B). Quan um yield o PSII pho ochemis y in
ligh -adap ed s a e (ΦPSII) o bo h K326 and Xiao eng 8 ini ially dec eased on shi om da k o bo h
low and high ligh , ollowed by an inc ease wi h inc easing ime (Fig. 3C and D). While he ini ial
dec ease was mo e p onounced upon ansi ion o high ligh han o low ligh , he ollowing inc ease was
highe upon ansi ion o low ligh han o high ligh . Ne e heless, no ob ious di e ences we e de ec ed
in changes o ΦPSII be ween he wo a ie ies unde same ligh in ensi y (Fig. 3C and D).
Mo e de ailed explo a ion o dependence o he s eady-s a e alues o he NPQ pa ame e s, ΦPSII,
and o he ela i e PSII elec on anspo a e (ETR) on in ensi ies o illumina ion by cons an
pho osyn he ic ac i e adia ion (PAR) is p esen ed in Fig. 4. The e is no signi ican di e ence in ETR
and ΦPSII be ween K326 and Xiao eng 8 a ie ies du ing exposu e o an inc easing amoun o cons an
PAR (Fig. 4A and 4B) and he same is ue o he NPQ pa ame e a PAR le els < 600 μmol pho ons m-
2 s-1 (Fig. 4C). Howe e , a highe s eady s a e NPQ alues we e de ec ed a cons an PAR > 400 μmol
pho ons m-2 s-1 in Xiao eng 8 han hose o K326 (Fig. 4C).
Cha ac e is ics o NPQ and ΦPSII in luc ua ing ligh in K326 and Xiao eng 8
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To in es iga e he esponse o NPQ pa ame e o con inuously luc ua ing ligh , seedling lea es o bo h
wo obacco a ie ies we e exposed o al e na ing cycles o 180 s a high ligh and 180 s low ligh . As o
he da k- o-low ligh ansi ion, Xiao eng 8 displayed insigni ican ly lowe NPQ pa ame e alue
compa ed wi h hose o K326. A e ansi ions om low o high ligh o e e y al e na ing cycle, alue
o NPQ pa ame e o Xiao eng 8 inc eased apidly and eached a highe le el han hose o K326, and
his pa e n was also de ec ed du ing he es high ligh pe iod h oughou he en i e ligh cycle. A e
ansi ions om high o low ligh , he alue o NPQ pa ame e dec eased apidly and no ob ious change
we e de ec ed be ween K326 and Xiao eng 8 (Fig. 5A). In e es ingly, no ob ious change we e de ec ed
in ΦPSII be ween he wo plan s h oughou he en i e low-high ligh cycle (Fig. 5B). Du ing low ligh
pe iods, he alue o ΦPSII is ela i ely high. Howe e , when he ligh in ensi y inc eases o high ligh ,
he ΦPSII alue apidly dec eases.
Responses o xan hophyll cycle pigmen s in K326 and Xiao eng 8 o high ligh
In o de o unde lie he mechanisms egula ing hei abili y o dissipa e excess ligh ene gy unde high
ligh , esponse pa e n o xan hophyll cycle pigmen s in K326 and Xiao eng 8 seedlings we e analyzed
a e exposu e o high ligh (1000 μmol pho ons m-2 s-1). The esul s showed ha , no signi ican di e ence
we e de ec ed in he h ee pigmen s, and he DEPS s a e be ween he wo a ie ies in da k. A e exposu e
o high ligh , he con en o V in Xiao eng 8 dec eased apidly wi hin he i s 10 min and educed o hal
a mos a e 10 min. On he o he hand, he con en s o A, and Z we e inc eased a e ansi ion om
da k o high ligh (Fig. 6). The pa e n o changes o xan hophyll cycle pigmen s in K326 is simila o
ha o Xiao eng 8, excep ha he con en o V is lowe in Xiao eng 8 han in K326 in high ligh and
con en o A, Z, and DEPS is highe in Xiao eng 8 han in K326 in high ligh (Fig 6).
Al e ed xan hophyll cycle pigmen s a ec ed seedlings NPQ unde high ligh in ensi y
To u he in es iga e he ole o xan hophyll cycle pigmen s in egula ing plan s abili y o dissipa e
excess ene gy unde apidly luc ua ing ligh condi ions, mu an s wi h mal unc ional VDE o zeaxan hin-
epoxidase (ZEP), enzymes ca alyzing he de-epoxida ion o V ia A o Z and he epoxida ion o Z ia A
o V, espec i ely, we e analyzed unde a ious ligh condi ions and compa ed wi h he wild ype plan
(K326) as con ol. Lack o he wo enzymes has signi ican ly al e ed xan hophyll cycle pool size. In bo h
de and zep knockou mu an , he con en o V, A, Z, o al xan hophyll cycle pool size and DEPS we e
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7.
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Dis inc non-pho ochemical quenching cha ac e is ics unde cons an and luc ua ing ligh
condi ions: compa ison be ween local and impo ed obacco a ie ies
Hui Lyu1，†, Zhaoqi Tang2，†, Weijie Xu2, Dejun Kong3, Zhihong Wang3, Zhixiao Yang3, Jishun Zhang3,
Hongqi Wu4, Zili Wang3, Dusan Laza 5, Yingchao Lin3,*
1 School o Biological Science and Ag icul u e, Qiannan No mal Uni e si y o Na ionali ies, Duyun,
China
2 Shanghai Tobacco G oup Co., L d, Shanghai, China
3 Guizhou Academy o Tobacco Science, Guiyang, China
4 College o Tobacco Science, Guizhou Uni e si y,Guiyang, China
5 Depa men o Biophysics, Facul y o Science, Palacky Uni e si y, Olomouc, Czech Republic
* Co espondence: [email protected]
† These au ho s con ibu ed equally o his wo k.
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Figu e legends
Figu e 1. Ten-day a e age sunshine hou and 10-d a e age empe a u e om Ap il o Oc obe a
Qingzhen in 2023.
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Figu e 2. Compa ison o g ow h o K326 and Xiao eng 8 seedlings g own in he ield a e ansplan ing
o 20 d. (A) Pheno ype o K326 ( igh idge) and Xiao eng 8 (le idge) in he ield. (B o F) Lea
numbe (B), maximum lea a ea (C), plan heigh (D), plan esh weigh (E) and d y weigh (F) o
seedlings g own in he ield a e ansplan ing o 20 d in Qingzhen, 2022. Da a a e p esen ed as means
± S.E.. (n > 10 biological eplica es) and he di e en le e s indica e signi ican di e ences (P < 0.05).
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Figu e 3. Kine ics o NPQ (A and B) and ΦPSII (C and D) on ansi ion om da k o cons an low ligh
(70 µmol pho ons m-2 s-1, A and C) and cons an high ligh (1000 µmol pho ons m-2 s-1, B and D).
Measu emen s we e made a e 30 min da k adap a ion in he seedlings g own in he ield a e
ansplan ing o 20 d in Qingzhen, 2022. Da a a e he means ±S.E.. Whe e no isible, e o ba s a e
smalle han he symbols.
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Figu e 4. Ligh dependence o s eady-s a e chlo ophyll luo escence pa ame e s o he ela i e PSII
elec on anspo a e (ETR, A), quan um yield o PSII pho ochemis y in ligh -adap ed s a e (ΦPSII, B),
and he non-pho ochemical quenching pa ame e (NPQ, C) in seedlings g own in he ield a e
ansplan ing o 20 d in Qingzhen, 2022. Each poin ep esen s he mean o 5 measu emen s and e o
ba s indica e he S.E. and he as e isk in (C) indica e signi ican di e ences (P < 0.05).
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Figu e 5. Dynamics o NPQ pa ame e (A) and ΦPSII (B) o luc ua ing ligh condi ions simula ed by
ou cycles o 3 min low ligh (70 μmol pho ons m−2 s−1, g ay backg ound) and 3 min high ligh
(1000 μmol pho ons m−2 s−1, whi e backg ound) as indica ed. Measu emen s we e made a e 30 min
da k adap a ion in he seedlings g own in he ield a e ansplan ing o 20 d in Qingzhen, 2022. Da a
a e he means ± S.E. Whe e no isible, e o ba s a e smalle han he symbols.
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Figu e 6. Changes in he con en o iolaxan hin (A), an he axan hin (B), zeaxan hin (C), and he
xan hophyll cycle de-epoxida ion s a e (DEPS, D) du ing illumina ion wi h 1000 µmol pho ons m−2 s−1
in he younges ma u e lea es o K326 and Xiao eng 8 seedlings g own in he ield a e ansplan ing
o 20 d in Qingzhen, 2022. Fully expanded lea es o seedlings ha p io da k adap ed o 30 min we e
collec ed a e 0, 1 min, 5 min, 10 min, 20 min and 30 min o illumina ion a 1000 µmol pho ons m-2 s-1.
Da a ep esen means ± S.E. o n=5 indi idual plan s. I no shown, S.E. is smalle han symbol.
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Figu e 7. (A) and (B), Gene a ion o mu a ions in VDE (LOC104218706, A) and ZEP (LOC104218557,
B) by CRISPR–Cas9, using a single-guide RNA. Sequences o VDE and ZEP in wild- ype (WT) and de
and zep mu an plan s a e shown. The dele ion is indica ed by a dashed line. (C-F), Con en s o
iolaxan hin (C), an he axan hin (D), zeaxan hin (E), and he xan hophyll cycle de-epoxida ion s a e
(DEPS, F) in 60-day-old wild ype (K326), de and zep knockou mu an plan s. (G) and (H), Dynamics
o NPQ pa ame e (G) and ΦPSII (H) o luc ua ing ligh condi ions simula ed by ou cycles o 3 min
low ligh (70 μmol pho ons m−2 s−1, g ay ba s) and 3 min high ligh (1000 μmol pho ons m−2 s−1, whi e
ba s) as indica ed in K326, de and zep knockou mu an plan s g own unde cons an ligh (1000
μmol pho ons m−2 s−1). Seedlings ha we e 45-day-old we e mo ed o g ow h chambe wi h a 16/8
ligh /da k cycle and a cons an ligh in ensi y o 1000 µmol pho ons m-2s-1. Fo (H) and (I), measu emen s
we e made a e 30 min da k adap a ion in mo e han i e ully indi idual plans. Da a a e he means
±S.E.. Whe e no isible, e o ba s a e smalle han he symbols, he di e en le e s in (C-F) indica e
signi ican di e ences (P < 0.05).
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