Ci a ion: Soengas, P.; Madloo, P.;
Lema, M. Spec al Re lec ance
Indexes Re eal Di e ences in he
Physiological S a us o B assica
ole acea wi h Con as ing
Glucosinola e Con en unde Bio ic
S ess. Plan s 2023,12, 2698. h ps://
doi.o g/10.3390/plan s12142698
Academic Edi o : Bo Sun
Recei ed: 12 June 2023
Re ised: 6 July 2023
Accep ed: 16 July 2023
Published: 19 July 2023
Copy igh : © 2023 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
plan s
A icle
Spec al Re lec ance Indexes Re eal Di e ences in he
Physiological S a us o B assica ole acea wi h Con as ing
Glucosinola e Con en unde Bio ic S ess
Pila Soengas * , Pa i Madloo and Ma ga i a Lema
G oup o Gene ics, B eeding and Biochemis y o B assicas, Misión Biológica de Galicia, Spanish Council o
Scien i ic Resea ch (MBG-CSIC), 36143 Pon e ed a, Spain; pa ib [email p o ec ed] (P.M.);
[email p o ec ed] (M.L.)
*Co espondence: [email p o ec ed]
Abs ac : B assica species p oduce glucosinola es, a speci ic g oup o seconda y me aboli es p esen
in he B assicaceae amily wi h an ibac e ial and an i ungal p ope ies. The employmen o imp o ed
a ie ies o speci ic glucosinola es would educe he p oduc ion losses caused by pa hogen a ack.
Howe e , he consequences o he inc emen in hese seconda y me aboli es in he plan a e un-
known. In his wo k, we u ilized e lec ance indexes o es how he physiological s a us o B asica
ole acea plan s changes depending on hei cons i u i e con en o glucosinola es unde nons ess ul
condi ions and unde he a ack o he bac e ia Xan homonas campes is p . campes is and he ungus
Scle o inia scle o io um. The modi ica ion in he con en o glucosinola es had consequences in he esis-
ance o bo h nec o ophic pa hogens, and in se e al physiological aspec s o he plan s. By inc easing
he con en in sinig in and glucob assicin, plan s dec ease pho osyn hesis e iciency (PR531, F
F
m
),
biomass p oduc ion (CHL-NDVI, SR), pigmen con en (SIPI, NPQI, RE), and senescence (YI) and
inc ease hei wa e con en (WI900). These a iables may ha e a nega i e impac in he p oduc i i y
o c ops in an ag icul u al en i onmen . Howe e , when plan s a e subjec ed o he a ack o bo h
nec o ophic pa hogens, an inc emen o sinig in and glucob assicin con e s an adap a i e ad an age
o he plan s, which compensa es o he decay o physiological pa ame e s.
Keywo ds:
plan seconda y me aboli es; Xan homonas campes is p . campes is;Scle o inia scle o io um;
iso hiocyana es; g ow h–immuni y adeo
1. In oduc ion
Glucosinola es (GSLs) a e seconda y me aboli es ound in 16 bo anical amilies o
dico yledonous angiospe ms, mainly o he o de o B assicales, and a e pa icula ly p e-
dominan in he B assicaceae amily [
1
]. They a e de i ed om aminoacids and hey can
be classi ied in o h ee chemical classes, depending on he side chain o hei p ecu so
amino acid: alipha ics wi h me hionine, alanine, aline, leucine, and isoleucine as p ecu -
so s; a oma ics de i ed om phenylalanine o y osine and indolics wi h yp ophan as a
p ecu so [2].
GSLs a e s o ed in plan cells in a non-ac i a ed o m. Upon cellula dis up ion caused
by inju ies, pes s, and nec o ophic pa hogens, my osinase enzymes come in o con ac wi h
GSLs and ca alyze hei hyd olysis in o a ious hyd oly ic p oduc s, which ha e an imi-
c obial e ec agains a b oad ange o plan pa hogens. The e ec o hyd oli ic p oduc s
depends on hei chemical s uc u e and on he plan pa hogen [
3
–
6
]. The GSLs-my osinase
sys em is pa o he immune sys em o B assicaceae plan s, including A abidopsis haliana
and B assica c ops [
7
–
10
]. GSLs a e cons i u i ely syn he ized and s o ed in plan cells
and ac mainly as phy oan icipins, al hough hey can be induced by pa hogen in ec ion
unc ioning in his case, as phy oalexins [3,4].
Plan s 2023,12, 2698. h ps://doi.o g/10.3390/plan s12142698 h ps://www.mdpi.com/jou nal/plan s
Plan s 2023,12, 2698 2 o 12
P e iously, we used mass selec ion o de elop a se o B assica ole acea a . acephala L.
(kale) popula ions di e ing in he con en o wo GSLs, he alipha ics sinig in (2-p openyl,
SIN) and he indolic glucob assicin (3-indolylme hyl, GBS) [
11
], ob aining ou popula ions,
wi h high (HSIN and HGBS) and low con en (LSIN and LGBS) o he a ge GSLs. The
esis ance o hese ou popula ions ha e been p e iously es ed agains wo B assica
pa hogens: he ungus Scle o inia scle o io um (Ss) and he bac e ia Xan homonas campes is
p . campes is (Xcc) [
8
]. Resul s showed ha he e ec s o GSLs we e dependen on he
pa hogen and he ype o GSL. Thus, he alipha ic SIN was inhibi o y o in ec ion by Ss and
he indolic GBS was inhibi o y o in ec ion by Xcc [8].
The employmen o imp o ed a ie ies o speci ic GSLs would educe he yield and
economic losses caused by he a ack o pa hogens. Howe e , we do no know how he
selec ion a ec s o he pa ame e s in he plan and i s po en ial impac on p oduc i i y.
Al hough high cons i u i e con en o de ensi e me aboli es may con e an ad an age o
plan s subjec ed o pa hogen a ack, hei syn hesis is esou ce-demanding and may in u n
become a disad an age unde non-s ess ul condi ions [
12
]. Cons i u i e de ense esponses
may educe he g ow h o plan s due in pa o he unnecessa y di e sion o ene gy ese es
in he absence o s ess [
12
]. Inc eased exp ession o de ense ai s consumes me abolic
esou ces a he cos o g ow h. Fo example, he A abidopsis R-gene RPM1 cos s 9% o
he yield when plan s a e no unde a ack om he pa hogen o which his gene con e s
esis ance [
13
]. Mo eo e , g ow h and de ense a e nega i ely egula ed no only h ough
me abolic consump ion bu also h ough he an agonism o de ensi e phy oho mones,
such as jasmonic acid and salicylic acid [14].
Theo e ically, GSL p oduc ion can inc ease pho osyn he ic equi emen s in A. haliana
by a leas 15% [
15
]. This cos associa ed wi h he p oduc ion o GSLs may impac o he
p ocesses o he plan and a ec i ness and g ow h. De ense-associa ed cos s may be
pa icula ly impo an in c ops o he same amily, since i can educe hei p oduc ion
and economic alue. The s udy o he balance be ween g ow h and de ense should be
conside ed be o e eleasing new imp o ed a ie ies wi h enhanced GSL con en .
Plan e lec ance is in luenced by lea su ace p ope ies and in e nal s uc u e, as well
as by he concen a ion and dis ibu ion o biochemical componen s, such as chlo ophyll
and ca o enoid pigmen s. The e o e, analysis o e lec ance can be used o assess bo h he
biomass and he physiological s a us o a plan [
16
]. In his wo k, we u ilized e lec ance
measu emen s o es how he physiological s a us o B. ole acea plan s changes depending
on hei cons i u i e con en o GSLs unde non-s ess ul condi ions and when challenged
by pa hogen a ack. We also discuss he conce ns in b eeding o esis ance o pa hogens.
2. Resul s
Lea e lec ance was measu ed and eco ded in plan s wi h con as ing con en o
GSLs unde con ol condi ions and inocula ed wi h Xcc and Ss pa hogens a di e en imes
pos inocula ion. Resul ing p o iles e ealed speci ic spec al pa e ns o popula ions
and ea men s (Supplemen a y Figu es S1–S4). Thus, we compu ed indexes based on
e lec ance o compa e he pe o mance o plan s di e ing in GSL con en and o compa e
be ween ea men s. The esul s o each compa ison a e shown below.
Gene ally speaking, he popula ion HGBS showed lowe index alues han LGBS
agains ime, al hough di e ences we e no always signi ican (Figu e 1). The e we e no
many signi ican di e ences be ween HSIN and LSIN (Figu e 1), al hough, as i happens
wi h GBS popula ions, HSIN ended o ha e lowe alues in he indexes han LSIN. The
excep ion is he index WI900, which was highe in HGBS and HSIN han in LGBS and
LSIN, espec i ely (Figu e 1). Plan s dec ease hei alues in he di e en indexes agains
ime, wi h he excep ion o YI and SIPI (Figu e 1), ela ed o he yellowing and senescence
o lea es, which inc eases agains ime.
Compa isons be ween con ol and inocula ed plan s a e shown a he ime when
in ec ion eached i s maximum le el: 21 days pos inocula ion in Xcc expe imen and 4 days
pos inocula ion in Ss expe imen . In he Xcc expe imen , HGBS showed less damage han
Plan s 2023,12, 2698 3 o 12
LGBS and LSIN showed less damage han HSIN (Figu e 2A). In he Ss expe imen , HSIN
was signi ican ly less damaged han LSIN (Figu e 2B).
Plan s 2023, 12, x FOR PEER REVIEW 3 o 13
Compa isons be ween con ol and inocula ed plan s a e shown a he ime when
in ec ion eached i s maximum le el: 21 days pos inocula ion in Xcc expe imen and 4
days pos inocula ion in Ss expe imen . In he Xcc expe imen , HGBS showed less dam-
age han LGBS and LSIN showed less damage han HSIN (Figu e 2A). In he Ss expe i-
men , HSIN was signi ican ly less damaged han LSIN (Figu e 2B).
Figu e 1. Spide plo s showing he a e aged e lec ance indexes and luo escence o con ol plan s
du ing ou weeks in wo popula ions di e ing in hei con en o GBS (HGBS and LGBS) and in
wo popula ions di e ing in hei con en o SIN (HSIN and LSIN). To ep esen he indexes in he
same scale, alues we e s anda dized agains ime by sub ac ing he a e age and di iding by he
s anda d e o . As e isks ep esen signi ican di e ences be ween HGBS and LGBS (black) and
be ween HSIN and LSIN ( ed) a p ≤ 0.05.
Figu e 1.
Spide plo s showing he a e aged e lec ance indexes and luo escence o con ol plan s
du ing ou weeks in wo popula ions di e ing in hei con en o GBS (HGBS and LGBS) and in
wo popula ions di e ing in hei con en o SIN (HSIN and LSIN). To ep esen he indexes in he
same scale, alues we e s anda dized agains ime by sub ac ing he a e age and di iding by he
s anda d e o . As e isks ep esen signi ican di e ences be ween HGBS and LGBS (black) and
be ween HSIN and LSIN ( ed) a p≤0.05.
Plan s 2023, 12, x FOR PEER REVIEW 4 o 13
Figu e 2. Lesion diame e o inocula ed plan s: (A) Xan homonas campes is p . campes is measu ed
21 days pos inocula ion; (B) Scle o inia scle o io um measu ed 4 days pos inocula ion. As e isks
ep esen signi ican di e ences be ween popula ions a p ≤ 0.05.
Con ols (non-inocula ed lea es) o HGBS and LGBS popula ions showed highe
alues o indexes F Fm, PR531, CHL-NDVI, SR, and RE han he espec i e inocula ed
popula ions wi h Xcc and lowe alues o SIPI and YI, al hough hese di e ences we e
no always signi ican (Figu e 3A). A simila end was obse ed when we compa e HSIN
and LSIN con ols wi h inocula ed popula ions wi h Xcc, al hough signi ican di e ences
we e ound mainly in LSIN compa isons (Figu e 3B).
In he Ss expe imen , HGBS con ol only di e ed signi ican ly om inocula ed
plan s o index WI900 (Figu e 3C). Con ol o LGBS showed signi ican ly lowe alues o
PR531, CHL-NDVI, SR, SIPI, and YI han inocula ed plan s. Wi h espec o HSIN and
LSIN popula ions, indexes ela ed o biomass and pho osyn hesis a e highe in con ol
plan s compa ed o inocula ed ones, al hough di e ences we e only signi ican o F Fm
and PR531 in HSIN and RE in LSIN. Those indexes ela ed o he deg ada ion o pig-
men s and yellowness ended o be highe in inocula ed plan s, al hough di e ences
we e only signi ican o YI in LSIN (Figu e 3D).
Figu e 2.
Lesion diame e o inocula ed plan s: (
A
)Xan homonas campes is p . campes is measu ed
21 days pos inocula ion; (
B
)Scle o inia scle o io um measu ed 4 days pos inocula ion. As e isks
ep esen signi ican di e ences be ween popula ions a p≤0.05.
Plan s 2023,12, 2698 4 o 12
Con ols (non-inocula ed lea es) o HGBS and LGBS popula ions showed highe
alues o indexes F
F
m
, PR531, CHL-NDVI, SR, and RE han he espec i e inocula ed
popula ions wi h Xcc and lowe alues o SIPI and YI, al hough hese di e ences we e no
always signi ican (Figu e 3A). A simila end was obse ed when we compa e HSIN and
LSIN con ols wi h inocula ed popula ions wi h Xcc, al hough signi ican di e ences we e
ound mainly in LSIN compa isons (Figu e 3B).
Plan s 2023, 12, x FOR PEER REVIEW 5 o 13
Figu e 3. Spide plo s showing he a e aged e lec ance indexes and luo escence o con ols and
inocula ed plan s o wo popula ions di e ing in hei con en o GBS (HGBS and LGBS) and SIN
(HSIN and LSIN) wi h pa hogens Xan homonas campes is p . campes is a e 21 days pos inocula-
ion (A,B) and Scle o inia scle o io um a e 4 days pos inocula ion (C,D). To ep esen he indexes
in he same scale, alues we e s anda dized agains ime by sub ac ing he a e age and di iding
by he s anda d e o . As e isks ep esen signi ican di e ences be ween he high GSL con en
popula ion and i s espec i e con ol (black) and be ween he low GSL con en popula ion and i s
espec i e con ol ( ed) a p ≤ 0.05.
In he compa ison o popula ions wi h high and low con en o GSLs a e being
challenged wi h pa hogens, we ound ha he e a e no many signi ican di e ences
be ween HGBS and LGBS agains ime a e being inocula ed wi h Xcc, on he con a y o
wha happened wi h he con ols (Figu e 4). LSIN showed, in gene al, highe alues in
he indexes a 7 and 14 d pos inocula ion han HSIN when bo h popula ions we e inoc-
ula ed wi h Xcc, wi h he excep ion o he index YI, which was highe in HSIN han in
LSIN (Figu e 4).
Figu e 3.
Spide plo s showing he a e aged e lec ance indexes and luo escence o con ols and
inocula ed plan s o wo popula ions di e ing in hei con en o GBS (HGBS and LGBS) and SIN
(HSIN and LSIN) wi h pa hogens Xan homonas campes is p . campes is a e 21 days pos inocula ion
(
A
,
B
) and Scle o inia scle o io um a e 4 days pos inocula ion (
C
,
D
). To ep esen he indexes in he
same scale, alues we e s anda dized agains ime by sub ac ing he a e age and di iding by he
s anda d e o . As e isks ep esen signi ican di e ences be ween he high GSL con en popula ion
and i s espec i e con ol (black) and be ween he low GSL con en popula ion and i s espec i e
con ol ( ed) a p≤0.05.
In he Ss expe imen , HGBS con ol only di e ed signi ican ly om inocula ed plan s
o index WI900 (Figu e 3C). Con ol o LGBS showed signi ican ly lowe alues o PR531,
CHL-NDVI, SR, SIPI, and YI han inocula ed plan s. Wi h espec o HSIN and LSIN
popula ions, indexes ela ed o biomass and pho osyn hesis a e highe in con ol plan s
compa ed o inocula ed ones, al hough di e ences we e only signi ican o F
F
m
and
PR531 in HSIN and RE in LSIN. Those indexes ela ed o he deg ada ion o pigmen s
and yellowness ended o be highe in inocula ed plan s, al hough di e ences we e only
signi ican o YI in LSIN (Figu e 3D).
In he compa ison o popula ions wi h high and low con en o GSLs a e being
challenged wi h pa hogens, we ound ha he e a e no many signi ican di e ences
be ween HGBS and LGBS agains ime a e being inocula ed wi h Xcc, on he con a y o
Plan s 2023,12, 2698 5 o 12
wha happened wi h he con ols (Figu e 4). LSIN showed, in gene al, highe alues in he
indexes a 7 and 14 d pos inocula ion han HSIN when bo h popula ions we e inocula ed
wi h Xcc, wi h he excep ion o he index YI, which was highe in HSIN han in LSIN
(Figu e 4).
Plan s 2023, 12, x FOR PEER REVIEW 6 o 13
Figu e 4. Spide plo s showing he a e aged e lec ance indexes and luo escence o inocula ed
plan s wi h Xan homonas campes is p . campes is du ing ou weeks in wo popula ions di e ing in
hei con en o GBS (HGBS and LGBS) and in wo popula ions di e ing in hei con en o SIN
(HSIN,] and LSIN). To ep esen he indexes in he same scale, alues we e s anda dized agains
ime by sub ac ing he a e age and di iding by he s anda d e o . As e isks ep esen signi ican
di e ences be ween HGBS and LGBS (black) and be ween HSIN and LSIN ( ed) a p ≤ 0.05.
When HGBS and LGBS we e inocula ed wi h Ss, LGBS showed highe alues o
mos o he indexes agains ime, excep YI, which was consis en ly and signi ican ly
highe in HGBS (Figu e 5). A 4 d pos inocula ion, HSIN showed highe alues o PR531,
CHL550, and CHL-NDVI han LSIN (Figu e 5).
Figu e 4.
Spide plo s showing he a e aged e lec ance indexes and luo escence o inocula ed plan s
wi h Xan homonas campes is p . campes is du ing ou weeks in wo popula ions di e ing in hei
con en o GBS (HGBS and LGBS) and in wo popula ions di e ing in hei con en o SIN (HSIN
and LSIN). To ep esen he indexes in he same scale, alues we e s anda dized agains ime by
sub ac ing he a e age and di iding by he s anda d e o . As e isks ep esen signi ican di e ences
be ween HGBS and LGBS (black) and be ween HSIN and LSIN ( ed) a p≤0.05.
When HGBS and LGBS we e inocula ed wi h Ss, LGBS showed highe alues o mos
o he indexes agains ime, excep YI, which was consis en ly and signi ican ly highe in
HGBS (Figu e 5). A 4 d pos inocula ion, HSIN showed highe alues o PR531, CHL550,
and CHL-NDVI han LSIN (Figu e 5).

Plan s 2023,12, 2698 6 o 12
Plan s 2023, 12, x FOR PEER REVIEW 7 o 13
Figu e 5. Spide plo s showing he a e aged e lec ance indexes and luo escence o inocula ed
plan s wi h Scle o inia scle o io um du ing i e days pos inocula ion in wo popula ions di e ing in
hei con en o GBS (HGBS and LGBS) and in wo popula ions di e ing in hei con en o SIN
(HSIN and LSIN). To ep esen he indexes in he same scale, alues we e s anda dized agains
ime by sub ac ing he a e age and di iding by he s anda d e o . As e isks ep esen signi ican
di e ences be ween HGBS and LGBS (black) and be ween HSIN and LSIN ( ed) a p ≤ 0.05.
3. Discussion
Lea e lec ance is a complex phenomenon dependen on biochemical and biophys-
ical p ope ies o he canopy lea es, which in u n a e a ec ed by g ow h condi ions and
diseases [17]. Pho osyn he ic pigmen s (chlo ophylls and ca o enoids) abso b ligh in he
isible spec um (400–700 nm). In he nea in a ed domain (700–1300 nm) e lec ance is
in luenced by he s uc u e o he lea and wa e con en , while in he middle in a ed
egion (1300–3000 nm) he a iabili y o e lec ance is linked o wa e con en and he
composi ion o lea chemicals [16].
In ou expe imen s we ha e ound ha high and low GSL con en popula ions
di e ed in hei spec al pa e ns. Following [18], he abso bance a 425 nm is co ela ed
o o al GSL con en in B assica napus, while [19] ound ha he spec al e lec ance in
se e al bands be ween 742 and 1000 nm is s ongly co ela ed o indi idual GSL con en
in kale (B. ole acea). O he di e ences in he spec al e lec ance be ween popula ions wi h
con as ing GSL con en e lec di e ences in hei igo , pho osyn hesis, o wa e con-
en . We will discuss in he nex sec ion how di e ences in he cons i u i e con en o
GSLs and he applica ion o bio ic s esso s modula e indexes based on e lec ance in he
in a ed egion o he spec um and i s ela ionship o he o e all physiological s a us o
he plan s.
Based on e lec ance indexes and in he F /Fm, LGBS has mo e biomass, i is mo e
pho osyn he ically e icien , i has less wa e con en , and less deg ada ion o pigmen s
han HGBS. The same end is obse ed when we compa e LSIN and HSIN. The e o e,
modi ica ion o GSL con en a ec s plan physiological s a us. GSLs a e pa o he de-
ense sys em o B assicaeae plan s. Howe e , hey can also in e e e wi h o he p ocesses
in he plan , al hough mechanisms go e ning his in e ac ion a e no clea . Some e i-
Figu e 5.
Spide plo s showing he a e aged e lec ance indexes and luo escence o inocula ed plan s
wi h Scle o inia scle o io um du ing i e days pos inocula ion in wo popula ions di e ing in hei
con en o GBS (HGBS and LGBS) and in wo popula ions di e ing in hei con en o SIN (HSIN
and LSIN). To ep esen he indexes in he same scale, alues we e s anda dized agains ime by
sub ac ing he a e age and di iding by he s anda d e o . As e isks ep esen signi ican di e ences
be ween HGBS and LGBS (black) and be ween HSIN and LSIN ( ed) a p≤0.05.
3. Discussion
Lea e lec ance is a complex phenomenon dependen on biochemical and biophysical
p ope ies o he canopy lea es, which in u n a e a ec ed by g ow h condi ions and
diseases [
17
]. Pho osyn he ic pigmen s (chlo ophylls and ca o enoids) abso b ligh in he
isible spec um (400–700 nm). In he nea in a ed domain (700–1300 nm) e lec ance is
in luenced by he s uc u e o he lea and wa e con en , while in he middle in a ed
egion (1300–3000 nm) he a iabili y o e lec ance is linked o wa e con en and he
composi ion o lea chemicals [16].
In ou expe imen s we ha e ound ha high and low GSL con en popula ions di e ed
in hei spec al pa e ns. Following [
18
], he abso bance a 425 nm is co ela ed o o al
GSL con en in B assica napus, while [
19
] ound ha he spec al e lec ance in se e al bands
be ween 742 and 1000 nm is s ongly co ela ed o indi idual GSL con en in kale (B. ole -
acea). O he di e ences in he spec al e lec ance be ween popula ions wi h con as ing
GSL con en e lec di e ences in hei igo , pho osyn hesis, o wa e con en . We will
discuss in he nex sec ion how di e ences in he cons i u i e con en o GSLs and he
applica ion o bio ic s esso s modula e indexes based on e lec ance in he in a ed egion
o he spec um and i s ela ionship o he o e all physiological s a us o he plan s.
Based on e lec ance indexes and in he F
/F
m
, LGBS has mo e biomass, i is mo e
pho osyn he ically e icien , i has less wa e con en , and less deg ada ion o pigmen s han
HGBS. The same end is obse ed when we compa e LSIN and HSIN. The e o e, modi ica-
ion o GSL con en a ec s plan physiological s a us. GSLs a e pa o he de ense sys em
o B assicaeae plan s. Howe e , hey can also in e e e wi h o he p ocesses in he plan ,
al hough mechanisms go e ning his in e ac ion a e no clea . Some e idences sugges
Plan s 2023,12, 2698 7 o 12
ha he e is a c oss- alk o he biosyn he ic GSL pa hway wi h he ho mone me abolism,
s oma al ape u e, he ci cadian clock, oo g ow h, biomass, and lowe ing [20–27].
In his wo k, HGBS and HSIN showed highe alues in he wa e index compa ed
o LGBS and LSIN du ing he i s 14 days o he expe imen . P obably, hese di e ences
a e ela ed o he s oma al closu e p omo ed by a highe con en in GSLs. Re e ence [
27
]
ound ha gg1 mu an s, de icien in he my osinase ha ca alyzes he p oduc ion o
iso hiocyana es (ITCs) om GSLs, we e hyposensi i e o abscisic acid (ABA) inhibi ion o
gua d cell inwa d K
+
channels and s oma al opening, e ealing ha he GSL–my osinase
sys em is equi ed o key ABA esponses o gua d cells. Mo eo e , he addi ion o SIN in
B. ole acea exposed o salini y s ess can egula e aquapo ins and wa e anspo [
25
]. GSLs
hemsel es do no su ice o inhibi channel ac i i y, sugges ing ha i is he hyd olyzed
p oduc s o he same ha e oke ion channel inhibi ion [
27
]. Ag eeing wi h his, he
exogenous applica ion o allyl-ITC, a deg ada ion p oduc om SIN, induced s oma al
closu e in A abidopsis haliana ia he p oduc ion o eac i e oxygen species, ni ic oxide,
and an inc ease in cy osolic Ca2+ [28].
Mo eo e , he in e ac ion o he GSL–my osinase sys em wi h ABA signaling could
also modi y he senescence o lea es, since ABA posi i ely egula es his p ocess [
29
].
Inc eases in he ela i e concen a ion o ca o enoids wi h espec o chlo ophyll a e o en
obse ed when plan s a e subjec ed o s ess and in senescing lea es [
16
]. Indexes YI and
SIPI a e based on his assump ion. In ou expe imen s, hey inc ease wi h he age o he lea
as is expec ed and dec ease in inc emen s in he cons i u i e con en o GSLs. The e o e,
high GSLs con en would p omo e an inc ease in wa e con en and delay in senescence.
Ano he ema kable esul om ou expe imen s is ha inc emen s in he GBS con en
in lea es lead o a dec ease in pho osyn hesis and biomass accumula ion. I is possible
ha his e ec is d i en by he hyd oly ic p oduc s o he GSL. The biomass o A. haliana
oo s dec eases a e he ex e nal addi ion o indol-3-ca binol, he main hyd oly ic p oduc
om GBS, in he lea es by inhibi ing auxin signaling h ough binding o he i 1 auxin
ecep o [
23
,
30
]. On he con a y, [
31
] e alua ed he ag onomic pe o mance o HGBS and
LGBS in di e en en i onmen s and analyzed hei me abolomic p o ile, inding ha HGBS
had a signi ican inc ease in esh and d y olia weigh compa ed o LGBS. Re e ence [
31
]
hypo hesized ha he be e ag onomic pe o mance o HGBS could be ela ed o an indi ec
inc ease in la onoids which can ac as g ow h egula o s. Ou expe imen s we e ca ied
ou wi h seedlings, unde g eenhouse condi ions, whe eas he expe imen s o [
31
] we e
conduc ed in ield condi ions wi h adul plan s exposed o he a ack o pes s, pa hogens,
and abio ic s esses. The e o e, i is possible ha , in ield condi ions, plan s om HGBS
ha e an ad an age o e hose om LGBS, which ha e o induce he syn hesis o mo e
seconda y me aboli es and, hus, eloca e esou ces om he p ima y me abolism.
Plan s om he HSIN popula ion also end o ha e lowe pho osyn hesis and biomass
indexes han LSIN. Re e ence [
20
] ound ha exogenous SIN applica ion lead o a ia ions
in biomass in A. haliana ha we e dependen on he suga concen a ion and on he en-
dogenous GSL con en o he plan . In his way, plan biomass accumula ion was nega i ely
co ela ed wi h he a io o me hyl hioalkyl/me hylsul inylalkyl GSL and posi i ely co -
ela ed wi h o al alipha ic GSL accumula ion [
20
]. The e ec o SIN on biomass may be
caused by i s hyd olysis by my osinases, since he addi ion o allyl-ITC caused g ow h
inhibi ion in A. haliana [
26
]. As was men ioned be o e, ITC ea men s lead o s oma al
closu e and his could also be ela ed o he loss o biomass.
In summa y, inc emen s in he cons i u i e con en o indi idual GSLs lead o a de-
c ease in pho osyn hesis e iciency, biomass p oduc ion, and senescence and o an inc emen
in wa e con en . This e ec was independen o he chemical class o GSLs, al hough
he esponses we e s onge in GBS geno ypes. The physiological e ec s o GSLs may be
d i en by hei hyd oly ic p oduc s, which could ake place a e punc u ing he lea es in
con ol plan s. The ela ionship o he amoun o GSLs wi h he physiological s a us o he
plan may espond o a g ow h–de ense adeo , whe e esou ces a e sen o he immune
sys em a he han o g ow h.
Plan s 2023,12, 2698 8 o 12
To es his hypo hesis, we challenged he ou geno ypes wi h nec o ophic pa hogens.
We ound di e ences in he spec al e lec ance be ween con ol and inocula ed plan s in
bo h expe imen s (Supplemen a y Figu es S1–S4). The physiological pe o mance o popu-
la ions is comp omised by hei in e ac ion wi h su ounding pa hogens. In ac , indexes
based on e lec ance ha e been employed o s udy he in ec ion caused by Xan homonas
species in ange ine [
32
] and ice [
33
] plan s. Spec al indexes we e e ec i e in he ea ly
de ec ion o X. campes is in b occoli plan s unde di e en clima ic condi ions [
17
]. In
addi ion, spec al cha ac e is ics ( anging om 1000 o 2500 nm) we e success ully used
o disc imina e lea es inocula ed wi h Al e na ia dauci om he o he h ee species o Al-
e na ia as well as om con ol plan s [
34
]. As expec ed, he con ols showed highe indexes
o pho osyn hesis and biomass, and lowe indexes o senescence han hei espec i e
inocula ed popula ions. Su p isingly, he LGBS popula ion does no show his end when
inocula ed wi h Ss.
Di e ences obse ed be ween he con ols o HGBS and LGBS disappea ed when
bo h popula ions we e inocula ed wi h Xcc. HGBS was signi ican ly less damaged han
LGBS. Taking his in o ma ion in o accoun , we sugges ha high GBS con en con e s a
de ensi e ad an age ha balances he in es men in his de ensi e compound and he
physiological s a us o he plan . Ag eeing wi h his heo y, he high con en o SIN does
no p o ec he plan agains Xcc; he e o e, LSIN was mo e esis an han HSIN and i s
physiological s a us was be e . The same balance be ween g ow h and de ense is obse ed
in Ss expe imen . HSIN pe o med be e o physiological pa ame e s han LSIN, p obably
due o hei highe esis ance o Ss. Ag eeing wi h ou esul s, [
35
] ound an associa ion
be ween GSL con en and inc eased seed p oduc ion when p eda o s we e p esen in
A. haliana. Howe e , he cos o his p oduc ion was a i ness de ec when p eda o s we e
absen [
36
]. Employing a lux balance analysis, [
15
] ound ha GSL p oduc ion can inc ease
pho osyn he ic equi emen s by a leas 15% in A. haliana. The e o e, an imp o emen in
GSL con en has associa ed cos s when plan s a e no subjec ed o an a ack by pa hogens,
bu cos s a e balanced by he plan unde bio ic s ess.
4. Ma e ials and Me hods
4.1. Pa hogens and Plan Cul u e
A se o popula ions o B. ole acea a . acephala L. (kale) di e ing in he con en o
wo GSLs, he alipha ic SIN (HSIN and LSIN) and he indolic GBS (HGBS and LGBS),
we e employed. The plan popula ions we e ob ained om a mass selec ion p og am a
he Misión Biológica de Galicia, Spanish Council o Scien i ic Resea ch (MBG-CSIC) [
11
].
Fo he expe imen s, popula ions we e g own in a g eenhouse wi h a 14 h pho ope iod, a
day–nigh mean empe a u e o 24/18
◦
C and 70% ela i e humidi y. Plan s we e g own in
po s con aining 2.5 L o pea (G amo lo GmbH & Co. KG P oduk ion, Vech a, Ge many).
The isola e o Xcc ace (s ain HRI3811, synonymous wi h PHW1205) was o iginally
collec ed om B. ole acea in he Uni ed S a es and was p o ided by Joana Vicen e (Wa wick
HRI, Wellesbou ne, UK). The Ss isola e MBG-Ss2 was p o ided by MBG-CSIC. The o iginal
isola e was collec ed in Janua y 2008 om a na u ally in ec ed plan o B assica napus in
an expe imen al ield a MBG. To main ain pa hogens agains ime, hey we e subcul u ed
pe iodically in PDA medium. Cul u es o Xcc we e incuba ed a 30
◦
C and cul u es o Ss
we e incuba ed wi h a 14 h pho ope iod, a day–nigh mean empe a u e o 24/18 ◦C.
4.2. Inocula ion and Physiological E alua ion unde Con ol and Bio ic S ess Condi ions
Two di e en expe imen s we e pe o med. In he i s one, plan s we e inocula ed
wi h Xcc and in he second one wi h Ss. The expe imen al design was he same in bo h
expe imen s. One hund ed and wen y plan s om each geno ype (HGBS, LGBS, HSIN,
and LSIN) we e employed. Since he p og ess o he disease a ies conside able depending
on he pa hogen, plan s inocula ed wi h Xcc we e moni o ed du ing ou weeks and plan s
inocula ed wi h Ss du ing i e days. In bo h expe imen s, plan s we e di ided in o wo
Plan s 2023,12, 2698 9 o 12
se s o wen y plan s each; one was inocula ed wi h he pa hogen and he o he ecei ed no
ea men . Plan s wi hin each ea men we e a anged in h ee epe i ions.
One se o plan s was inocula ed and he o he one ecei ed no ea men . In he
i s expe imen , ou weeks a e sowing he plan s, esh bac e ial colonies o Xcc we e
sub-cul u ed on Pe i dishes con aining PDA and incuba ed a 32
◦
C o 24 h in he da k.
A loop o bac e ia was ans e ed o nu ien b o h and shaken o e nigh a 150 pm
and 30
◦
C in he da k. The cul u e was dilu ed in s e ile wa e o a concen a ion o
5×108c u·mL−1
. F esh bac e ial inoculum was injec ed in o h ee di e en poin s o each
lea . In he second expe imen , esh mycelium o Ss was ob ained h ough ou ine ans e
o mycelial–aga plugs om he ma gin o a colony o Ss g owing on PDA medium. The
cul u es we e incuba ed o e a 14 h pho ope iod, wi h a day/nigh mean empe a u e
o 24/18
◦
C. Aga plugs con aining he ad ancing edge o ungal mycelia we e used as
he inoculum sou ce. Lea es we e inocula ed by placing one aga plug wi h he ungal
inoculum on he uppe side o he lea . In bo h expe imen s, he second younges lea
coun ing om he apex was inocula ed wi h pa hogen (inocula ed plan s) o wi h wa e
(con ol plan s). Inocula ed lea es we e pho og aphed. Then, a lesion a ea was ob ained
using ImageJ so wa e (Ve sion 1.51n).
The physiological s a us o he plan s in bo h expe imen s was assessed using e-
lec ance measu emen s and employing a UniSpec SC spec o adiome e (PP Sys ems,
Ha e hill, MA, USA). In bo h expe imen s, measu emen s we e conduc ed in he inocu-
la ed lea o each plan . Then, wi h he ou pu ma ix, se e al indexes we e compu ed
using AVICOL .6 So wa e. Based on e lec ance, di e en indexes ha e been p oposed
o check he physiological s a us o he plan and unc ional p ocesses. In his wo k, we
ha e employed indexes o es ima e biomass, wa e and pigmen con en , and pho osyn-
hesis e iciency. Wa e con en was es ima ed by employing WI900 o he ‘wa e index’.
Biomass was es ima ed using CHL-NDVI o he a io be ween e lec ance a 750 and 550 nm
(R750/R550) and NDVI index ‘no malized di e ence ege a ion index’ and wi h SR o
‘simple a io’ [
16
]. Pigmen con en was measu ed using SIPI, o he ‘s uc u al indepen-
den pigmen index’ ha measu es ca o enoids/chlo ophyll a io and NPQI ‘no malized
phaeophy iniza ion index’ ha measu es he chlo ophyll deg ada ion. The YI ‘yellowness
index’ [
37
] measu es he chlo osis o lea es in s essed plan s. RE o ‘ ed edge’ is an es ima e
o chlo ophyll con en . PR531 o he ‘pho ochemical adia ion index’ indica es he pho o-
syn he ic adia ion-use e iciency [
16
]. Chlo ophyll luo escence yield was measu ed in he
same lea es as e lec ance, using a po able Chlo ophyll luo ome e (Mini-PAM; Heinz
Walz, GmbH, E el ich, Ge many). Fluo escen ansience was induced using ed ligh
o 3000
µ
moL m
−2·
s
−1
p o ided by an a ay o 3 ligh -emi ing diodes (peak a 660 nm)
using plan s da k-adap ed o 20 min. Pa o he ligh ene gy abso bed by chlo ophyll
o pho osyn hesis is los as hea o luo escence; hus, measu emen s in luo escence
emissions can be employed o assess chlo ophyll e iciency. Then, we employed he F
F
m
a io based on luo escence and he PR531 index based on in a ed e lec ance as a p oxy o
pho osyn hesis e iciency.
4.3. S a is ical Analysis
Analyses o a iance we e pe o med using he GLM p ocedu e o S a is ical Analysis
So wa e (SAS, Ins i u e Inc., Ca y, NC, USA). An indi idual analysis o a iance was
compu ed by expe imen (Xcc,Ss) and ime. Compa isons o means be ween ea men s
we e conduc ed using a S uden ’s es a he 0.05 le el o p obabili y. Th ee di e en
analyses we e pe o med. Fi s o all, we compa ed he physiological s a us be ween he
con ols o he high and low GSLs popula ions. Then, we compa ed he physiological
s a us o he con ols s. inocula ed popula ions. Finally, we compa ed he pe o mance
be ween popula ions wi h high and low GSL con en inocula ed wi h Xcc o Ss. Popula-
ions and ea men s we e conside ed ixed ac o s, whe eas eplica ions we e conside ed
andom ac o s.