Freezing induces an increase in leaf spectral transmittance of forest understorey and alpine forbs

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Pho ochemical & Pho obiological Sciences (2022) 21:997–1009
h ps://doi.o g/10.1007/s43630-022-00189-0
ORIGINAL PAPERS
F eezing induces aninc ease inlea spec al ansmi ance o  o es
unde s o ey andalpine o bs
TwinkleSolanki1· JoséIgnacioGa cíaPlazaola2· T.Ma hewRobson1 · Bea izFe nándezMa ín3
Recei ed: 11 Oc obe 2021 / Accep ed: 10 Feb ua y 2022 / Published online: 28 Feb ua y 2022
© The Au ho (s) 2022
Abs ac
E e g een plan s g owing a high la i udes o high ele a ions may expe ience eezing e en s in hei pho osyn he ic issues.
F eezing e en s can ha e physical and physiological e ec s on he lea es which al e lea op ical p ope ies a ec ing emo e
and p oximal sensing pa ame e s. We oze lea es o six alpine plan species (Soldanella alpina, Ranunculus kuep e i, Luzula
nu ans, Gen iana acaulis, Geum mon anum, and Cen au ea uni lo a) and h ee e e g een o es unde s o ey species (Hepa ica
nobilis, F aga ia esca and Oxalis ace osella), and assessed hei spec al ansmi ance and op ically measu ed pigmen s,
as well as pho ochemical e iciency o pho osys em II (PSII) as an indica o o eezing damage. Upon eezing, lea es o
all he species ansmi ed mo e pho osyn he ically ac i e adia ion (PAR) and some species had inc eased ul a iole -A
(UV-A) ansmi ance. These di e ences we e less p onounced in alpine han in unde s o ey species, which may be ela ed
o highe chlo ophyll deg ada ion, isible as educed lea chlo ophyll con en upon eezing in he la e species. Among
hese unde s o ey o bs, he hin lea es o O. ace osella displayed he la ges educ ion in chlo ophyll (−79%). This s udy
p o ides insigh s in o how eezing changes he lea op ical p ope ies o wild plan s which could be used o se a baseline
o upscaling op ical e lec ance da a om emo e sensing. Changes in lea ansmi ance may also se e oindica e pho o-
syn he ic su iciency and physiological ole ance o eezing e en s, bu expe imen al esea chis equi ed o es ablish his
unc ional associa ion.
T. Ma hew Robson and Bea iz Fe nándezMa ín sha ed senio
au ho ship.
* T. Ma hew Robson
ma hew[email p o ec ed]
1 O ganismal andE olu iona y Biology (OEB), Viikki
Plan Science Cen e (ViPS), Facul y o Biological
andEn i onmen al Sciences, Uni e si y o Helsinki,
00014Helsinki, Finland
2 Depa men o Plan Biology andEcology, Uni e si y
o  heBasque Coun y (UPV/EHU), Ba io Sa iena s/n,
48940Leioa, Spain
3 Depa men o Bo any, Ecology andPlan Physiology,
Uni e si y o La Laguna (ULL), 38200Tene i e, Spain
998 Pho ochemical & Pho obiological Sciences (2022) 21:997–1009
1 3
G aphical abs ac
Keywo ds F ozen lea es· Ex eme clima ic e en s· Lea op ical p ope ies· Spec al e lec ance· Pho op o ec ion· Lea
pigmen s
1 In oduc ion
Pe ennial plan s in alpine en i onmen s and in empe a e
unde s o y o es s a e a na u al isk o eezing damage.
This isk may be highes in ea ly sp ing, when pho osyn-
he ic issues eme ge om unde insula ing snow co e ,
be o e a aining ull os ha diness [1, 2]. Exposu e o
such eezing e en s can damage buds and lea es, leading
o educed lowe ing o ui ing, and ul ima ely mo ali y
in bo h alpine plan s [3–6] and o es unde s o ey species
[4, 7, 8]. Plan s ha e e ol ed a ious adap a ions agains
eezing bu ne e heless eezing esis ance a ies g ea ly
ac oss species, anging om hose ha a e e y empe a u e
sensi i e o some alpine and bo eal species ha e en su -
i e ex eme eezing empe a u es [2, 9, 10]. Canopy co e
pa ially bu e s unde s o ey plan s om he sha p d ops in
empe a u e expe ienced in open en i onmen s o bo eal and
alpine egions [11], bu al e na ely canopy in e cep ion and
he e ogenei y can educe snow co e lea ing g ound eg-
e a ion exposed o eezing empe a u es. Alpine plan s a e
ypically be e adap ed o eezing condi ions han o es
unde s o ey species; howe e , e en hey can also su e sig-
ni ican loss o biomass, ecundi y and ul ima ely compe i-
i e abili y ollowing eezing e en s [12]. F eezing esis -
ance can also a y h ough he seasons, among popula ions
wi hin a single species, and e en among plan o gans and
de elopmen al s ages [2, 13–15]. F eezing de ini ely ig-
ge s physiological, biochemical and physical al e a ions in
he lea es, ega dless he capabili y o issues o ole a e
sub-ze o empe a u es.
Sha p d ops in empe a u e can occu du ing sp ing in
alpine zones and o es unde s o ies a high ele a ion o la i-
ude [11]. These sudden luc ua ions in empe a u e du ing
pe iods when ull physiological unc ion is s ill e u ning o
o e win e ing lea es o en causes chlo ophyll deg ada ion
[16] and s imula es accumula ion o an ioxidan pigmen s,
such as an hocyanins, which amelio a e s ess a low empe -
a u es [17, 18], and o he la onoids which ha e addi ional
oles including UV-sc eening, os p o ec ion and he bi o y
de ence [17, 19–21]. F eezing, also gene a es changes in he
pigmen composi ion o lea es: o ins ance, in os - ole an
lea es eezing induces he de-epoxida ion o xan hophyll
cycle ca o enoids in ol ed in pho op o ec ion [22], e en in
da kness [23–25].
In addi ion o he changes in pigmen s, eezing em-
pe a u es may cause s uc u al changes o he lea and lea
su ace a ec ing hei op ical p ope ies. On he one hand,
physiological and ana omical esponses o a oid lea dam-
age a cold empe a u es, including acclima ion o he cell
memb ane composi ion and cell s uc u e, may modi y lea
op ical p ope ies a ime-scales o days o seasons [26].
On he o he hand, ice accumula ion in apoplas ic spaces,
999Pho ochemical & Pho obiological Sciences (2022) 21:997–1009
1 3
cellula cy o hysis, o changes o he cu icle ha e associa ed
e ec s on he sca e ing o adia ion in he lea [26–29]. In
ac , ice op ical p ope ies g ea ly di e o hose om liquid
wa e , and i s bi e ingence has ecen ly been used o loca e
ice c ys als wi hin lea es and buds h ough e lec ed ligh
mic oscopy [30].
Ve y ew app oaches a e a ailable o de ec exo he ms,
o isualize ice o ma ion and p opaga ion he mally, o
o e alua e ice alloca ion wi hin plan issues in o de o
add ess he mechanisms and consequences o plan eez-
ing [27, 31–35]. Howe e , by examining he ansmi ance
o adia ion in ozen lea es and compa ing i wi h ha o
esh lea es we can in e i s unc ional signi icance, as well
as p o iding in o ma ion on e lec ance ha can be scaled-up
o in e p e emo e sensing da a [36] wi h b oad applica ions
in c op and ecosys em science.
All p e ious poin s conside ed, we aimed o de e mine
he changes induced by eezing in he spec al e lec ance
and ansmi ance o lea es om alpine and unde s o ey
plan s, spanning he ul a iole (UV; 280–400nm), PAR
(400–700nm) and a - ed (700–900nm wa eleng hs)
egions o he spec um. In o de o p o ide insigh s in o
physiological p ocesses po en ially associa ed wi h changes
in he spec a, we assessed he pho osyn he ic yield o pho-
osys em II (F /Fm) and op ically measu ed lea pigmen s,
be o e and upon eezing. To examine he consis ency o
hese esponses be ween alpine meadow species and bo eal
o es unde s o ey o bs, we es ed se e al plan species
om each en i onmen in sp ing soon a e snowmel . We
expec ed alpine plan species, om a habi a whe e luc u-
a ing empe a u es and high i adiances a e common, o be
be e acclima ed o eezing, including ha ing mo e pho-
op o ec i e pigmen s. Because o his, we also expec ed
alpine species o display a smalle inc ease in lea spec al
ansmi ance and smalle d op in pho osyn he ic yield ol-
lowing eezing compa ed o he o es unde s o ey species.
2 Ma e ials andme hods
2.1 Collec ion si es, plan ma e ial
andexpe imen al design
Lea es o six o he mos p ominen sp ing ime alpine spe-
cies [37] we e sampled om an alpine meadow in he sub-
ni al zone o he wes e n F ench Alps adjacen o he S a-
ion Alpin Joseph Fou ie (SAJF) bo anical ga den a he
Col du Lau a e (45.0359°N, 6.4052°E) a 2150m a.s.l,
on 28 h May 2019. These species we e: Soldanella alpina
(alpine snowbell), Ranunculus kuep e i (Py enean bu e -
cup), Luzula nu ans (wood ush), Gen iana acaulis ( um-
pe gen ian), Geum mon anum (alpine a ens) and Cen au-
ea uni lo a (alpine knapweed). Likewise, lea es o h ee
common o b species we e sampled om he unde s o ey o
a mixed bo eal o es [38], domina ed by Be ula pendulaand
Be ula pubescens wi h some Picea abies ees, a Lammi
Biological S a ion LBS, cen al sou he n Finland (61.05°N,
E 25.04°E) a 130–135m a.s.l on 15 h June 2020. These
unde s o ey o bs we e: Hepa ica nobilis (li e wo ), F a-
ga ia esca (wild s awbe y) and Oxalis ace osella (wood
so el). In he alpine meadow, only lea es om he p e ious
yea ha had su i ed unde snow co e h ough he win e
we e sampled, while in he unde s o ey new lea es we e
also sampled. All lea es we e collec ed close o sola noon
be ween 12:00 and 14:00 o con ol o diu nal a ia ion in
lea ai s. A pa allel se o lea es we e sampled concu en ly
om which lea a ea, esh weigh and d y mass (a e d y-
ing o a cons an weigh a 60°C) we e eco ded. Fo alpine
species, 6 g oups o 5 lea disks we e weighed, whe eas
among unde s o ey species, 20–25 en i e lea es we e used,
o calcula e speci ic lea a ea (SLA: lea o disk a ea di ided
by d y mass) and lea wa e con en (LWC: one minus d y
mass o e esh weigh ) [39] (Table1). Mean daily em-
pe a u e, p ecipi a ion and ela i e humidi y in he alpine
meadow du ing he expe imen we e 5.0°C, 0mm and 73%
(SAJF wea he s a ion, sou ce: h ps:// www. da is- me eo.
com/ Van a ge- P o2. php) and in he unde s o ey hey we e
20.6°C, 0mm and 80% (LBS, sou ce: h ps:// en. ilma ie ee
nlai os. i/) (Fig.1).
2.2 F eezing ea men s andchlo ophyll
luo escence measu emen s
One ma u e lea was ha es ed om h ee- o- ou di e en
plan s o each species (and lea -age class, whe e applicable)
g owing na u ally in he alpine meadow and he o es unde -
s o ey., Immedia ely a e sampling lea eswe e aken o he
lab o op ical measu emen s. Du ing he ans e pe iod
om ield o lab, o abou 10–15min, he esh lea es we e
kep in sealed plas ic bags in a cool box. All h ee se s o
measu emen s: op ical p ope ies, chlo ophyll luo escence
and lea pigmen s we e made on e e y lea . To eeze he
lea es, hey we e placed ho izon ally la be ween wo shee s
o pape owel, o ensu e ha hey didn’ c inkle on eezing,
e u ned o sealed plas ic bags and ans e ed o he eeze
a − 18°C o 24h in da kness, di ec ly ollowing oom-
empe a u e measu emen s on esh lea es. This same se
o lea es we e emeasu ed when ozen, keeping he lea es
insi u in he eeze o ensu e ha hey emained ozen
and a he same empe a u e h oughou all he di e en
measu emen s.
Indices o lea chlo ophyll con en , and epide mal la o-
noid glycosides ( la onols in dico s., la ones in monoco s.)
and epide mal an hocyanins we e measu ed once om he
adaxial side o each lea wi h a Dualex Scien i ic + (Fo ce-
A, Pa is Sud, F ance). Maximum quan um yield o PSII
1000 Pho ochemical & Pho obiological Sciences (2022) 21:997–1009
1 3
(F /Fm), as indica o o pho osyn he ic capaci y, was meas-
u ed wi h a Fluo Pen (PSI, D áso , Czechia) ollowing
30min o da k acclima ion using lea clips. We made a se
o measu emen s o lea spec al ansmi ance and e lec-
ance o adia ion o each o he h ee lea es pe alpine
species o ou lea es pe unde s o ey species and lea -age
class. These measu emen s we e epea ed on he same lea es
once ozen, excep o e lec ance o alpine species which
was no logis ically possible. Following hese measu emen s
and 30-min da k-adap a ion, F /Fm (mini-PAM, Heinz-Walz,
E el ich, Ge many) was eco ded as a means o indi ec ly
assessing ue eezing wi hin he issues o he same se o
ozen lea es. The ozen lea es o alpine species we e oo
agile o allow Dualex and e lec ance measu emen s be o e
lea es de os ed.
2.3 Measu ing andp ocessing o lea op ical
p ope ies
Lea spec al ansmi ance and e lec ance we e measu ed
ac oss he spec al egion (250–892nm), encompassing
all he wa eleng hs o ambien UV-B and UV-A adia ion,
and isible and a - ed ligh ha plan s na u ally ecei e in
sunligh . Re lec ance o adia ion om he adaxial (uppe )
side and ansmi ance o he abaxial (lowe ) side o one
lea om h ee- o- ou eplica e plan s pe species and age
class we e measu ed wi h a dual in eg a ing-sphe e sys-
em, Jaz Spec o-Clip (Ocean Op ics, Dundane, FL, USA).
These measu emen s we e all aken om he same pa
o he esh and ozen lea es selec ed o all o he op i-
cal measu emen s, a oiding he mid ib whe e possible.
The Jaz Spec o-clip comp ises se e al modules includ-
ing a dual spec ome e , a pulse xenon ligh sou ce, da a
p ocessing uni and ba e y. The wo in eg a ing sphe es
in he sys em collec ligh ansmi ed hough he lea as
well as e lec ed ligh . The Jaz modula spec ome e was
used as a s andalone o op ical measu emen s, he ig-
ge a e was se o 10ms wi h a hold-o ime and ig-
ge delay o 1ms, and he spec ome e in eg a ion ime
was se o 1000ms. Each lea ’s ansmi ance/ e lec ance
spec um was an a e age o six consecu i e indi idual
eco dings o spec a wi hou bo h boxca -smoo hing and
non-linea i y de ec ion. Measu emen s o each lea ook
c4min., du ing which ime he e was no no iceable end
o sugges any sho - e m ime-dependen a ia ion in op i-
cal p ope ies. The lash a e was se a 200Hz (o one
lash e e y 5ms) wi h an in ensi y o 400 Vol s. Each
lea ansmi ance and e lec ance spec um measu ed on a
sample (S), was ma ched wi h a da k (D) and e e ence (R)
measu emen , using black and whi e Spec alon di use-
e lec ance e e ence a ge s (WS-1-SL, Ocean Op ics).
Table 1 Lea mo phological ai s om he alpine and unde s o ey species used in he expe imen
Lea es o plan s g owing in he expe imen al si es a he ime o measu emen we e used o calcula e speci ic lea a ea (SLA), lea a ea (LA) and
lea wa e con en (LWC), as he mean (± 1 s anda d de ia ion) o 20–25 lea es o unde s o ey plan s, and o six g oups o i e lea disks om
alpine species
Lea p oduc ion Lea ana omy Lea shape T ichomes Speci ic
lea a ea
(mm2 mg−1) ¥
Lea a ea
(cm2) ¥Lea wa e
con en (%)
Alpine species
Cen au ea
uni lo a Pe ennial Do si en al Na ow lanceo-
la e
Dense sho
ough hai
15.647 ± 1.532 2.69 ± 0.75 76.78 ± 1.01
Gen iana
acualis Annual/pe ennial Do si en al Ellip ical o
lanceola e
Hai less 11.914 ± 1.405 10.16 ± 3.52 70.05 ± 2.10
Geum mon a-
num Pe ennial Do si en al Pinna e Densely Hai y 11.305 ± 0.805 5.87 ± 0.56 64.54 ± 0.64
Luzula nu ans Pe ennial Isobila e al Na ow lanceo-
la e
Hai y 18.752 ± 1.375 1.76 ± 0.17 81.07 ± 1.36
Ranunculus
kuep e i Pe ennial Do si en al Na ow lanceo-
la e
13.291 ± 1.372 3.82 ± 0.29 79.32 ± 0.06
Soldanella
alpina Pe ennial Do si en al Kidney Hai less 12.407 ± 0.77 3.59 ± 1.01 70.57 ± 1.14
Unde s o ey species
Hepa ica
nobilis Once annually Do si en al Loba e 3 lea le s Few hai s 25.64 ± 4.33 9.28 ± 3.23 71.49 ± 0.25
Oxalis ace o-
sella Sequen ially
g owing season
Isobila e al Palma e i olia e Few hai s 81.97 ± 13.16 5.53 ± 1.44 82.86 ± 2.59
F aga ia esca Dimo phic
sp ing/au umn
Do si en al Palma e 3
lea le s
Hai y 48.09 ± 4.54 14.82 ± 2.16 67.08 ± 2.52
1001Pho ochemical & Pho obiological Sciences (2022) 21:997–1009
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The ins umen calcula es he spec al ansmi ance (Tλ)
and e lec ance(Rλ) acco ding o Eq.1.
Pos -p ocessing o he aw spec a was done using he
Pho obiology sui e o packages in R [40], o de ec ou -o -
ange alues. A “lowess” unc ion was selec ed o smoo h
he spec um, a e es ing and compa ing hose smoo h
unc ions a ailable, educing he e ec o bad pixels wi hou
o e i ing he da a.
2.4 S a is ical analyses
Di e ences in he ansmi ance, e lec ance and abso p ance
o lea es we e compa ed in wo ways, ea ed ei he as con-
inuous spec a o as disc e e spec al egions calcula ed om
hese spec a. The mean o spec a ( om 3 o 4 lea es) o
(1)
T
𝜆∕R𝜆=
S−D
R−D
×
100,
a species, o eezing ea men , we e conside ed o di e
signi ican ly when hei 95% con idence bands we e non-
o e lapping, plo ed in he same igu e. Whe eas, species o
eezing- ela ed di e ences o disc e e spec al egions we e
es ed h ough ANOVA.
Two-way ANOVA was used o es whe he he spec al
ansmi ance in blue, g een, ed and a - ed (and likewise
F /Fm) di e ed among species, be o e s upon eezing. Di -
e ences in lea epide mal pigmen s ( la onols, an hocyanin
and chlo ophyll con en ) due o lea age, among unde s o ey
species, and be o e s upon eezing, we e es ed by a h ee-
way ANOVA, and among alpine species lea epide mal pig-
men s we e es using a single ac o ANOVA. Func ion glh
om ‘mul icomp’ package was used o es ima e wi hin-species
pai -wise compa isons and mul iple compa isons be ween
esh and ozen lea es.
Fig. 1 Time se ies o mean ai empe a u e measu ed a 10min in e -
al a bo h sampling loca ions; Lammi Biological S a ion, Finland
(unde s o ey species) and he Col du Lau a e , F ance (alpine spe-
cies). Inse plo s in blue boxes zoom in on he empe a u e on he day
when plan lea es we e sampled om bo h he loca ions

1002 Pho ochemical & Pho obiological Sciences (2022) 21:997–1009
1 3
3 Resul s anddiscussion
F eezing caused changes in he op ical p ope ies ( e lec-
ance and ansmi ance ac oss he UV, PAR and a - ed
spec um), as well as pigmen s (op ically measu ed la-
onols, an hocyanins and chlo ophylls) o ozen lea es
compa ed wi h esh lea es om species g owing in alpine
and o es unde s o ey en i onmen s. While a ew s ud-
ies ha e add essed spec al cha ac e iza ion o lea -inju y
induced a e eezing in c op plan s [36, 41] and in ees
[42], o he bes o ou knowledge, his is he i s s udy
examining he spec al ansmi ance and e lec ance o
ozen lea es in wild species. The esul s o his se ies o
measu emen s o lea op ical p ope ies, and associa ed
lea pigmen s in he same lea es, could p o ide insigh s
in o a ia ion in he amoun o ligh eaching he meso-
phyll upon eezing and se a baseline o emo e sensing
whe e such da a on lea ansmi ance and e lec ance o
ozen lea es in win e a e equi ed as inpu s o modeling
adia i e ans e .
3.1 F eezing inc eased ansmi ance o lea es
o bo halpine andunde s o ey species
The ansmi ance spec a o esh lea es om ou s udied
species ollowed cha ac e is ic pa e ns simila o hose p e-
iously epo ed o lea es om a a ie y o species [43,
44] (Fig.2). A ypical lea appea s g een o he human eye,
due o he s ong abso p ion by pho osyn he ic pigmen s
Fig. 2 Measu ed spec al e lec ance and ansmi ance and calcu-
la ed abso p ance (280–892 nm) o esh lea es p io o eezing
(black line) and o ozen lea es (g ay line). The blue band indi-
ca es he 95% con idence in e al a ound each spec um, o ma u e
lea es (solid line) and young lea es (dashed line). Whe e he bands
a ound spec a a e non-o e lapping, he di e ence be ween hem is
conside ed s a is ically signi ican . a Alpine species: Each spec um
is he mean o h ee lea es, wi h each lea ’s spec um composed o
six consecu i e spec a om a single posi ion on he lea . No e ha
e lec ance spec a we e no eco ded om ozen lea es excep o G.
mon anum. b Unde s o ey species: Each spec um is he mean spec a
om ou lea es collec ed om di e en loca ions in he unde s o ey
o he s and. Each lea ’s spec um is composed o six consecu i e
spec a om a single posi ion on he lea
1003Pho ochemical & Pho obiological Sciences (2022) 21:997–1009
1 3
o ligh a o he wa eleng hs in he isible spec um (Pho-
osyn he ically Ac i e Radia ion—PAR, 400–700nm).
A longe wa eleng hs, in he nea and sho -wa e in a-
ed (700–2500nm) g een lea es ypically ha e p omi-
nen ly highe e lec ance and ansmi ance, wi h e y li le
abso p ance; p ope ies linked o hei wa e con en and
s uc u al ea u es [45–49]. In ou expe imen , lea spec al
ansmi ance inc eased in esponse o eezing bu he size
o his e ec di e ed ac oss he spec um (Fig.3). O e all,
he inc ease in he pe cen age o adia ion ansmi ed was
la ges in he a - ed (inc easing by 4.1% in alpine sp and
7.6% in unde s o ey sp) and in he PAR (1.4% alpine sp and
4.5% unde s o ey sp); whe e speci ically he di e ence in
ansmi ance be ween esh and ozen lea es was g ea e in
he g een (500–600nm; 1.84% alpine sp and 7.74% unde -
s o ey sp) han he ed (600–700nm; 0.14% alpine sp and
5.45% unde s o ey sp) and blue (400–500nm; 0.40% alpine
sp and 1.85% unde s o ey sp) spec al egions (Figs.2 and
4, TableS5). O e all, ozen lea es also ansmi ed 0.60%
(alpine sp) and 0.24% (unde s o ey sp) mo e UV-A adia-
ion (315–400nm) han esh lea es o bo h he unde s o ey
and alpine species, bu his e ec was minimal in he UV-B
(280–315nm) because all lea es ansmi ed negligible adi-
a ion (< 0.1%) in his spec al egion, dec easing a sho e
wa eleng hs (Fig.2, TableS5).
The enhancemen o ansmi ance induced by eez-
ing was gene ally g ea e in he lea es o unde s o ey han
alpine species (Fig.2a, b). Howe e , in bo h en i onmen s
he e we e some species which de ia ed om his gene al
pa e n o esponse. E en once ozen, he lea es o mos
alpine species ( i e o he six species) we e opaque o UV-A
adia ion and blue ligh (Figs.2a, and 4), and L. nu ans only
ansmi ed less han 0.02% UV-A adia ion (Figs.2a and
4). Luzula nu ans was he only monoco among he species
ha we es ed, and had high SLA and LWC compa ed wi h
he o he alpine species (Table1), as well as a e y di e en
lea s uc u e; which may explain his di e ence in ans-
mi ance [50, 51]. A ypical monoco lea has a compac
palisade mesophyll o en e e ed o as chlo enchyma and
lacks spongy mesophyll; his causes ligh in e cep ion, sca -
e ing and ansmi ance o be e y di e en om species
wi h bo h spongy and palisade mesophyll cells [52]. Among
alpine species, only lea es o G. mon anum and R. keup e i
ansmi ed signi ican ly mo e g een ligh (inc easing by
2.7% and 4.8%) and a - ed ligh (inc easing by 6.5% and
11.3%) when ozen han esh (Fig.2a, TableS4); indica -
ing ha eezing had a g ea e impac on lea op ical p ope -
ies in hese species han in he o he alpine o bs. Among
he unde s o ey species, esh lea es o O. ace osella had
he highes spec al ansmi ance among he species we
measu ed; and likewise, once ozen, i s lea es ansmi ed
he mos spec al i adiance (Figs.2b, 4 and TableS4). I e-
spec i e o damage, he ac ha O. ace osella has e y hin
lea es [53–56] likely explains why hei ansmi ance was
highes , his con as s wi h H.nobilis which has hick lea es
h ough which e y li le adia ion is ansmi ed (Figs.2b
and 4, Table1) [54]. As well as lea s uc u e, speci ic lea
a ea and lea ana omy, di e ence in ansmi ance among
species may change due o acclima ion o he physical en i-
onmen , such as empe a u e luc ua ions and high inciden
i adiance, e.g., inducing pho o opin-media ed chlo oplas
s acking, whe eby chlo oplas s align agains he an iclinal
cell walls inc easing ligh ansmi ance h ough he lea
[55, 57]. The p ocesses leading o apoplas ic ice alloca ion,
o ganelle eo ganiza ion and o he ul as uc u al changes
upon eezing a e as ye poo ly unde s ood in plan lea es.
Howe e , ecen me hodological ad ances ha e enabled ice
accommoda ion wi hin lea c oss-sec ions o be isualized
[30], and o ul as uc u al e alua ion o ozen lea es by
ansmission elec on mic oscopy [58]. These echniques
appea o be p omising new ools o complemen he in e -
p e a ion o changes in spec al signa u es o ozen lea es
in he nea u u e.
No ably, he e lec ance o O. ace osella lea es in he
PAR inc eased when ozen, wi h he g een peak seemingly
shi ing owa ds longe wa eleng hs (Fig.2b). Lea abso b-
ance in his spec al egion is mainly a ibu able o chlo o-
phyll, hence his change may be he esul o cell lysis a ec -
ing he pH, deg ading chlo ophyll and p oducing pheophy in
[42]. I is also possible ha dis up ion o he memb ane
and cell s uc u e o he lea du ing eezing con ibu ed
o he changes eco ded in ansmi ance and e lec ance,
educing he sca e ing o adia ion by in e nal s uc u es,
Fig. 3 Maximum quan um yield o pho osys em II (PSII) pho ochem-
is y (F /Fm) o six alpine species o esh lea es p io o eezing
and o ozen lea es. Each poin is he mean ± 1 SE o ou lea es.
Signi ican di e ences be ween esh and ozen lea es o each spe-
cies a e indica ed as ollow: *p < 0.05, **p < 0.01, ***p < 0.001.
S. alpina among alpine species, and O. ace osella among all unde -
s o ey species, had signi ican ly lowe alue o F /Fm han he es
(p < 0.001)
1004 Pho ochemical & Pho obiological Sciences (2022) 21:997–1009
1 3
which would also esul in educed back-sca e ed adia ion
escaping he lea . In all h ee unde s o ey species, eezing
educed lea e lec ance in he a - ed egion. The amoun o
adia ion e lec ed in he a - ed is hough o be con olled
by su ace p ope ies, ai spaces and he in e nal s uc u e o
he lea [52]. Addi ionally, he phase ansi ion om wa e o
ice has been ound o displace e lec ance spec a o longe
wa eleng hs in he g een and a - ed egions du ing eezing
o oil-seed ape (B assica napus) lea es [36].
When exposed o eezing empe a u es and high i adi-
ance, lea es can su e se e e pho o-inhibi o y s ess and
damage o he pho osyn he ic appa a us. In eezing- ole an
species, his isk is e ec i ely coun e ac ed wi h pho op o-
ec i e mechanisms [59–61]. Ou expe imen , howe e , was
conduc ed in da kness o a oid pho oinhibi ion. F esh lea es
om he unde s o ey species showed high F /Fm alues o
0.82, and simila ly, esh lea es o he alpine species had
high F /Fm a 0.78 on a e age. Values o F /Fm sligh ly
below 0.8, such as hese, a e common unde op imal condi-
ions o alpine species gene ally [24, 25, 62]. Acclima ion
o pho osyn he ic appa a us om eezing- ole an species o
low empe a u es is usually accompanied by a down egula-
ion o p edawn F /Fm alues [23, 24]. In e es ingly, in ou
s udy, he F /Fm dep ession due o eezing was highe in
he unde s o ey han he alpine species. The g ea e sus-
cep ibili y o unde s o ey species o eezing damage may
ha e been exace ba ed because hey we e sampled la e
in o he g owing season han he alpine species, meaning
ha hey a e likely o ha e comple ely deha dened p io o
ou  eezing ea men [14, 63]. Amongs he unde s o ey
species, F /Fm was lowe ed mos by eezing in O. ace o-
sella (Fig.3) which was consis en wi h he la ges inc ease
in ansmi ance occu ing in his species, along wi h he
g ea es loss o chlo ophyll and lowes la onoid indices
Fig. 4 Spec al e lec ance, abso p ance and ansmi ance plo ed o
di e en spec al egions: UV-B, UV-A, blue, g een, ed and a - ed
o all unde s o ey and alpine plan species, compa ing esh lea es
p io o eezing agains ozen lea es. Spec al egions we e calcu-
la ed om he spec a plo ed in Fig.2A, B. Only ma u e lea es we e
used in his plo (Fig.2A, B)
1005Pho ochemical & Pho obiological Sciences (2022) 21:997–1009
1 3
(Figs.2b and 5). Amongs alpine species, S.alpina had bo h
he lowes F /Fm in esh lea es (0.72 ± 0.01), and he big-
ges dec ease in ozen lea es ( o 0.37 ± 0.04), compa ed
wi h F /Fm in o he alpine species which only declined o
abou 0.60 (Fig.3). While we did no ind a di e ence in
he ex en o which F /Fm in ma u e lea es, ha had o e -
win e ed, was dep essed by eezing compa ed wi h cu en
yea ’s lea es, p e ious s udies [64, 65] ha e epo ed young
lea es o alpine species o be mo e eezing sensi i e, since
ice o ma ion can mo e easily damage hei pho osyn he ic
appa a us han in ma u e lea es.
3.2 Changes upon eezing inop ically measu ed
lea pigmen s
Lea chlo ophyll, epide mal la onol and an hocyanin con-
en s we e measu ed op ically on he lea es o unde s o ey
species be o e and upon eezing, whe eas hese op ical ai s
we e only measu ed in esh lea es o alpine species. The
esh lea es o alpine species con ained highe epide mal la-
onoids as well as highe chlo ophyll con en han he esh
lea es o unde s o ey species (Fig.5a and b). This p esum-
ably is a consequence o he accumula iono hese pigmen s
in esponse o he highe sola adia ion, in pa icula UV-B
adia ion, ecei ed in he alpine en i onmen han in he
bo eal unde s o ey, and i s colde sp ing ime empe a u es
wi h la ge diu nal luc ua ions [38, 66], which s imula e
inc eased pho op o ec ion and chlo ophyll accumula ion [60,
66]. I is concei able ha excision o he esh lea es p io
o measu emen could a ec hei physiology and pigmen
alues p io o eezing, bu compa ison wi h o he a ached
lea es o hese species did no p o ide e idence o such an
e ec (unpublished da a).
Fo all he unde s o ey species, he index o epide mal
la onols was highe han ha o an hocyanins, bu hese
indices di e ed in esponse o eezing (Fig.5b and c). In
unde s o ey species, he epide mal an hocyanin index sig-
ni ican ly inc eased upon eezing in da kness (p < 0.001)
(Fig.5c). Howe e , he e was no pa allel inc ease in he
op ical index o UV-sc eening epide mal la onols upon
eezing in O.ace osella o F. esca, i only inc eased in
H.nobilis (Fig.5b). Wi hou a quan i a i e compa ison o
he me aboli es o esh and ozen lea es, we a e no able
o a ibu e hese inc eases in abso bance o changes in he
concen a ion o loca ion o hese classes o la onoid. E en
well-acclima ed alpine species such as Ramonda myconi
cease enzyma ic ac i i y a −15°C [24], so a mos hese
lea es would ha e been me abolically ac i e only as hei
empe a u e d opped p io o eezing, meaning he e would
ha e been li le oppo uni y o an hocyanin syn hesis.
In con as o la onols, he d op in op ically meas-
u ed lea chlo ophyll con en upon eezing di e ed
signi ican ly among species. Chlo ophyll con en was
Fig. 5 Lea chlo ophyll (a), and epide mal la onol (b) and an hocya-
nin (c) con en s o esh lea es o six alpine species (Lau a e ) be o e
eezing, and h ee unde s o ey o bs (Lammi) o esh lea es p io
o eezing ( ed poin s) and ozen lea es (blue poin s). Signi ican
di e ences be ween esh and ozen lea es o each species a e indi-
ca ed as black as e isks, and di e ences be ween ma u e and young
lea es as blue and ed as e isks: *p < 0.05, **p < 0.01, ***p < 0.001.
Mean alues wi h di e en le e s indica e signi ican di e ences
among unde s o ey species (uppe case) and alpine species (lowe
case). Each poin is he mean ± 1 SE o 3 and 4 lea es o alpine and
unde s o ey species, espec i ely. Whe e Dualex da a a e absen ,
lea es we e oo delica e o his measu emen when ozen