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Qualidurin
Diego DInstituto de
CCT-CONICE
a r t i c
Article histoReceived 24Received inAccepted 2Available on
Keywords:MitochondrRootSenescenceCarbohydraRespirationArabidopsis
1. Introd
The ttural dettheir devphysiologremobilizleaves to
tively stuas roots. Sies concesource oforgans [2particula
Mitoccarbon- aexchangeconstitut[4–6]. Th
∗ CorrespE-mail a
http://dx.do0168-9452/
Plant Science 258 (2017) 112–121
Contents lists available at ScienceDirect
Plant Science
j ourna l ho me pa g e: www.elsev ier .com/ locate /p lantsc i
tative and quantitative modifications of root mitochondriag senescence of above-ground parts of Arabidopis thaliana
arío Fanello ∗, Carlos Guillermo Bartoli, Juan José GuiametFisiología Vegetal (INFIVE), Facultades de Ciencias Agrarias y Forestales y de Ciencias Naturales y Museo, Universidad Nacional de La Plata,T La Plata, cc 327, 1900, La Plata, Argentina
l e i n f o
ry: August 2016
revised form 18 January 20171 January 2017
line 7 February 2017
ia
a b s t r a c t
This work studied modifications experienced by root mitochondria during whole plant senescence orunder light deprivation, using Arabidopsis thaliana plants with YFP tagged to mitochondria. During post-bolting development, root respiratory activity started to decline after aboveground organs (i.e., rosetteleaves) had senesced. This suggests that carbohydrate starvation may induce root senescence. Similarly,darkening the whole plant induced a decrease in respiration of roots. This was partially due to a decreasein the number of total mitochondria (YFP-labelled mitochondria) and most probably to a decrease inthe quantity of mitochondria with a developed inner membrane potential (19m, i.e., Mitotracker red-labelled mitochondria). Also, the lower amount of mitochondria with 19m compared to YFP-labelledmitochondria at 10d of whole darkened plant, suggests the presence of mitochondria in a “standby state”.
te starvation
thaliana
The experiments also suggest that small mitochondria made the main contribution to the respiratoryactivity that was lost during root senescence. Sugar supplementation partially restored the respirationof mitochondria after 10d of whole plant dark treatment. These results suggest that root senescence istriggered by carbohydrate starvation, with loss of 19m mitochondria and changes in mitochondrial sizedistribution.
© 2017 Elsevier B.V. All rights reserved.
tlinges tochonng leon- ptory aucti
bjectenedity (
s conight
easeabolidria
s det
uction
erm senescence refers to the functional and struc-erioration observed in plants during the final stages ofelopment [1]. Monocarpic senescence involves differentical processes occurring after flowering, including theation of mineral and organic compounds from senescinggrowing fruit and seeds [1]. Senescence has been exhaus-died in leaves and fruits but scarcely in other organs suchince much of the focus was on leaf senescence, most stud-
ntrated on chloroplasts since these organelles are the main carbon and nitrogen remobilized from leaves to other,3]. Knowledge about changes in other organelles, and
rly in mitochondria, is still very limited.hondria participate in primary metabolism involvingnd nitrogen-containing compounds. The synthesis and
manleavmitodurior npiraa redis sudarkactivstay
Ldecrmetchonroot
of ATP/ADP, redox compounds and carbon skeletonse the main functions carried out by plant mitochondriaese functions are considered critical for the proper dis-
onding author.ddress: [email protected] (D.D. Fanello).
in vitro cSome
morpholowere obs“enlargedZottini andria of M
i.org/10.1016/j.plantsci.2017.01.013© 2017 Elsevier B.V. All rights reserved.
of chloroplasts and translocation of nutrients from the other organs during senescence [7,8]. The number ofdria (calculated on the basis of cell volume) decreasesaf senescence, in either photosynthetic (i.e., mesophyll)hotosynthetic leaf cells (i.e., epidermal cells) [8]. The res-ctivity of senescent leaves decreases as a consequence of
on in the amount of mitochondria when the whole planted to dark conditions [8]. However, when only one leaf is
and the rest of the plant is kept under light, the specificnmol O2 min−1 mg−1 protein) of remaining mitochondriastant [8].deprivation abolishes photosynthesis, leading to a
of carbohydrate levels, which affects mitochondrialsm. The impact of decreased carbohydrate levels on mito-
was studied using different experimental models, such asached from the plant [9], plant cells in culture [10,11] andulture of apical root sections [12].evidences suggest an association between mitochondrialgy and carbohydrate starvation; spherical mitochondria
erved in cultured cells under normal conditions, whereas” mitochondria were predominant under starvation [11].d cowokers showed morphological changes in mitochon-edicago truncatula during different phases of protoplast
8 (201
culture [1stages of
obtained
lar metabmitochonbohydrat
Alteracell deathcess [15].of plant mSeveral wmorpho-ever, senPCD in aundergo
“bleb”, anto PCD [2
The mand qualital senescand seneto carboh
2. Mater
2.1. Plan
Arabidthe yellounder coin all expas descrient solutiMgSO4,
0.9 mM MpH 5.5–6in 200 mLent soluticontaininplaced ata growthdark) for
ural seneand day/nand kept
cut off at tsolution iFinally, tinutrient sbefore stwas repla15–20 lea
2.2. Indu
For wof Arabidand inflorcence wadarknesslight conplants in
with conantimyco
mg/l
on.
Mea
holem in
e at
genr tother
of
ge dspira170 m) we
SDS-
oots
l anae in
4 mMPMSged
ctedrding
mixHCl
captoples
lamidern
e m) non
7.4, 2 antib2; [2
USA). Aft
PBS a sected todielope
mM5% vternsuncte in e
set ais. In
higheller vr tha
Conf
resh
l fluo oil imesce
D.D. Fanello et al. / Plant Science 25
3]. These cells had giant mitochondria during the initialsenescence. In addition, different mitochondrial fractionsfrom sections of maize root apexes showed particu-olic and structural properties and a reduced amount ofdria with low density when roots were subjected to car-e starvation [14].tions in mitochondria were observed during programmed
(PCD) and might be important for the progress of this pro- However, there are few analysis focusing on the behavior
itochondria during senescence, and much less in roots.orks have studied the relationship between changes in
functional aspects of mitochondria and PCD [16–21]. How-escence of organs, such as leaves, appears to differ from
number of significant ways. Cells in senescing organsa gradual, orderly disassembly, the cytoplasm does notd senescence can be reversed in various ways, contrary2].ain objectives of this work were to study quantitativetative changes in root mitochondria during developmen-ence of above-ground organs of plants (mainly the leaves)scence induced by darkness, and to relate these changesydrate deprivation.
ial and methods
t material and growth conditions
opsis thaliana (Heyn.) ecotype Col-0, transformed withw fluorescent protein (YFP) targeted to mitochondriantrol of the 35S promoter (mt-YFP lines, [23]) was usederiments. Plants were grown in a hydroponic system
bed by Norén et al., with modifications [24]. The nutri-on consisted of 5.0 mM Ca(NO3)2, 5.0 mM KNO3, 2.0 mM1.0 mM KH2PO4, 20.0 mM FeNaEDTA, 5.0 mM H3BO3,nCl2, 0.8 mM ZnCl2, 0.3 mM CuSO4 and 0.01 mM Na2MoO4,
.5 as described by Leggett and Frere [25]. Seeds were sown pipette tips sealed at the base and filled with 50% nutri-on in 0.7% (w/v) agar. Tips were arranged in a closed rackg a wet paper towel to keep humidity high. The rack was
4 ◦C in darkness for 4 days. Then it was transferred to chamber, with a short-day photoperiod (10 h light/14 hdarkness assays and long days (16 h light/8 h dark) for nat-scence assays. Irradiance was 120 mmol m−2 s−1 (PPFD)ight temperature was 22/17 ◦C. Seeds were germinated
under these conditions for 10 days. Then pipette tips werehe base and placed with their bases submerged in nutrientn a plastic container sealed with plastic film for 2 weeks.ps were placed in individual pots containing 400 mL ofolution with air bubbling. Plants were grown for 4 weeks
arting the darkness incubation assays. Nutrient solutionced every 2 weeks (as the plants grew larger, more thanves, nutrient solution was replaced more frequently).
ction of senescence and glucose treatment
hole plant natural senescence experiments, the stagesopsis development were established according to rosetteescence growth progression. In other experiments, senes-s induced by transferring Arabidopsis plants to continuous
for 5 or 10 d, with control plants maintained under normal
250
erati
2.3.
Wsystetrodwerepapeas obasischanof re(70–after
2.4.
Runtimaderol,w/v
trifucolleaccowereTris–merSamacrywestlulos(w/vpH
cific(coxgen,[29]withwithjugaantibdeve(0.230.01WesGel fvalu(andto ththe
smalowe
2.5.
Ffoca60 xfluor
ditions. Glucose treatment was performed by incubatingnutrient solution with the addition of 0.1 M glucose (+glu),trol plants without glucose (−glu) added. An antibiotic-tic mixture consisting of 50 mg/l ampicillin [26] and
tial (19m(MT, Moing root
sections
7) 112–121 113
amphotericin [9] was applied to prevent microbe prolif-
surement of root respiration
plant root respiration was measured by placing the root an air-tight chamber equipped with a Clark type O2 elec-
25 ◦C (Hansatech, UK). Prior to the measurements, rootstly washed with deionized water and then blotted with
wels to remove excess water. Root respiration, as wellparameters (e.g., sugar content) was expressed on the
fresh weight because percentage of dry matter did noturing root ontogeny (data not shown). For measurementstion, the entire rosette or samples of the inflorescenceg fresh weight for 4 week-old plants, 200–400 mg there-
re placed in an air-tight chamber.
PAGE and inmunobloting
were harvested, frozen in liquid N2 and kept at −80 ◦Clysis. Extracts (200 mg root fresh weight/mL buffer) were0.1 M bicine buffer, pH 7.5, 1 mM EDTA, 10% v/v glyc-
cysteine, 1% w/v PVPP and protease inhibitors (0.004%F and 0.001% w/v Leupeptin). Homogenates were cen-at 1500g and 4 ◦C for 20 min and the supernatants were
for subsequent analysis. Soluble proteins were analyzed to the protocol described by Laemmli [27]. Supernatantsed (1/1 v/v) with sample buffer 2 x consisting of 125 mMpH 6.8, 10% (v/v) glycerol, 4% (w/v) SDS, 10% (v/v) 2-b-ethanol, 6 M urea, and 0.005% (w/v) bromophenol blue.
were loaded on a 2 mm thick, 12% (w/v) denaturing poly-e gel and electrophoresed at 20 mA gel−1 for 2.5 h. For
blotting, proteins were electro-transferred to a nitrocel-embrane at 200 mA for 1 h. Blots were blocked in 5%-fat dry milk dissolved in PBS (10 mM phosphate buffer,.7 mM KCl, and 137 mM NaCl) and probed with spe-odies for mitochondrial proteins: Cytochrome c oxidase
8]), cytochrome c (cytC; catalogue no. 554002, BD Pharmi-) and L-Galactone-1,4-lactone dehydrogenase (L-GalLDH;er washing the membrane three times for 10 min each
plus Tween 20 0.05% (w/v, PBS-T), blots were incubatedondary antibody (goat anti-rabbit or anti-mouse IgG con-o horseradish peroxidase, for polyclonal or monoclonals, respectively) for 1 h and washed with PBS-T. Blots wered by incubation in 10 mL of chemiluminescence mixture
coumaric acid, 1.2 mM luminol dissolved in DMSO and/v H2O2 in Tris-HCl buffer 0.1 M pH 8.5). SDS-PAGE and
blots were analyzed with the ImageJ software, Analyzeion (1.46r version). For protein quantitation, the highestach image (Gel or Western blot) was used as a references 1), and then all other bands in the same gel were referred
all cases the reference (=1) was the control replicate withst protein content, with other control replicates havingalues. For this reason, the average values for controls aren 1.
ocal laser microscopy and staining procedures
root samples were observed using an SP5 inverted con-rescence microscope (Leica, Mannheim, Germany) with a
mersion objective. Mitochondria were detected by YFPnce, and mitochondria with an inner membrane poten-
) [30,31] were observed with Mitotracker Red CMXRoslecular probes, USA). This dye was applied by incubat-sections in a 0.5 mM MT solution for 20 min; then, rootwere washed 3 times with deionized water and finally
114 8 (201
observedsettings wMT. Quanareas shounder th(version
ment of rof numbeparticle alocalizatiwhere: Moverlappportion oAnother
analysis.
2.6. Solub
Solublmethod [accordingsurementaccordingmethod 2
Stati
ata wresul
henalitalit
kal-W
esul
Root
itial and
scen plan
d outained
planol O2nific
Fig. 1. Postthaliana corat differentdata (roots,senescenceppw) Seconppw) Much
D.D. Fanello et al. / Plant Science 25
with the confocal laser microscope. Excitation/emissionere 514/524–550 nm for YFP and 543/596–680 nm for
titation of mitochondria density (i.e., number of discretewing YFP and/or MT signal per root section observed
e microscope) was carried out with the ImageJ software1.46r). The procedure included: selection and measure-oot area, determination of threshold level, quantificationr and size of discrete areas with YFP or MT signal (with thenalysis feature) and analyses of data in a spreadsheet. Co-on analysis was performed using Manders coefficient (M)1 (YFP → MT) corresponds to the proportion of YFP signal
ing with MT and M2 (MT → YFP) corresponds to the pro-f MT signal overlapping with YFP. The JACoP Plugin (JustCo-localization Plugin, [32]) was used for co-localization
le sugar and starch measurements
e sugar measurements were made with the Anthrone33] using an ethanol extraction from fresh root tissues
2.7.
Dand
(*). WNormNormKrus
3. R
3.1.
Intionseneafterdrierempost(mma sig
to the protocol described by Costa et al. [34]. Starch mea-s were performed in the pellet after ethanol extraction
to the protocol described by Rose et al. [35] (enzymatic).
3.2. Effec
The d(rosette a
-bolting senescence of plants grown under a 16:8 h light/dark photoperiod regime. a) Bresponding to 4, 5, 13, 17, 21 and 26 weeks post-planting (dry tissues were not measure
stages of development in Arabidopsis thaliana. Different letters indicate significant dif rosette leaves and inflorescence shoots). DW = dry weight, ppw = Post-planting weeks. D
of green tissues and post-planting weeks (ppw), as described below: 4 ppw) Initial boldary inflorescence shoots with flowers and siliques; rosette begins to senesce. 17 ppw)
of the inflorescence shoots dry. 26 ppw) Shoots almost completely dry. Suplementary Fi
7) 112–121
stical analysis
ere analyzed using the Student’s T-test and ANOVA teststs were considered as statistically different when P ≤ 0.05
variance (Levene test P ≥ 0.05) or normality (Skewnessy of Residuals, Kurtosis Normality of Residuals or Omnibusy of Residuals test P ≥ 0.05) were regarded as different, a
allis test was applied.
ts
respiration during plant development
experiments were carried out to study root respira-its changes during normal post-bolting development andce under a photoperiod of 16 h. Bolting started 4 weeksting. Plants lost their rosette leaves (i.e., they completely) after 13 weeks post planting, while inflorescence shoots
alive, and roots retained their biomass even 21 weeksting (Fig. 1a). Roots maintained their respiratory activityg−1 FW h−1) until late stages of plant development, with
ant drop only at 26 weeks (Fig. 1b).
ts of darkness on rosette and root biomass
ecrease in root respiration following the death of leavesnd cauline) might be a consequence of photoassimilate
iomass of roots, rosette leaves and inflorescence shoots of Arabidopsisd for rosette and inflorescence shoots biomass). b) Respiratory activity
ferences (ANOVA P ≤ 0.05) between sampling dates within each set ofevelopment stages were established according to grown progress and
ting. 5 ppw) Primary inflorescence shoot with flowers and siliques. 13 Proliferation of secondary inflorescence shoots; rosette totally dry. 21g. S1 provides images of plants.
D.D. Fanello et al. / Plant Science 258 (201
Fig. 2. Effects of light deprivation on the shoot and root biomass of plants subjectedto 5 or 10 days of darkness (5d or 10d, respectively) and control plants kept undera 10:14 h light/dark photoperiod regime. a) Root and b) Rosette. Empty bars repre-sent controls kept at light/dark regime, light gray bars represent 5d of darkness anddark gray baences (ANOof plants.
deprivatihydrate sof plantsprotracteon day 5respectiv
3.3. Resptreated pl
Root rwith planto mentio
respiratoindicatintreatmenchanges.
Similatein conttotal andplants weGalLDH.
3.4. Quan
Typicaareas shodecreaseobservedof darknchondriaa pronoudarknessshows thness do nmost of t(Fig. 5c).
An anaccordinging YFP a
creas5–1.5
dec5–1.
6b).
Starcleme
o detls of
h cot, eit
Fig. 3. Rootregime. Emindicate sig
rs represent 10d of darkness. Different letters indicate significant differ-VA P ≤ 0.05). FW = fresh weight. Suplementary Fig. S2 provides images
on. To test this hypothesis, and to mimic possible carbo-tarvation conditions during the post-bolting development, root respiration was measured in plants incubated ind darkness. Both roots and rosette leaves ceased to grow
after the initiation of the dark treatment (Fig. 2a and b,ely).
iratory activity and protein content in roots of darkants
a deof 0.largeof 0.(Fig.
3.5.
supp
TleveStarcmen
espiration decreased after 5d of dark treatment comparedts under normal light conditions (Fig. 3). It is importantn that plants maintained under light displayed a constant
sugars dement (Fian increa(Fig. 8). N
respiratory activity of plants subjected to 5 or 10 days of darkness (5d or 10d, respectivelpty bars represent controls kept at light/dark regime, light gray bars represent 5d of danificant differences (ANOVA P ≤ 0.05).
7) 112–121 115
ry activity during the 10d-period the experiments lastedg that differences observed were the consequence of darkt and not due to growing conditions or plant ontogenetic
r trends were observed for total and mitochondrial pro-ents (Fig. 4). The differences between 5d and 10d in both
mitochondrial protein content of roots from dark treatedre not significant (Student’s T-test P ≥ 0.05) except for L-
tification of mitochondria by confocal microscopy
l images of YFP and MT-labelled root mitochondria (i.e.,wing YFP and/or MT signal) are shown in Fig. 5a. A
in the abundance of mitochondria marked with YFP was in tangential optical planes of cortical root cells after 10 dess treatment (Fig. 5b). Similarly, the amount of mito-
detected with MT (mitochondria with a 19m) showednced decrease after 10 d in darkness, compared to 5 d in
and light controls (Fig. 5b). The co-localization analysisat most of the mitochondria remaining after 10 d in dark-ot maintain a 19m (i.e., M1 coefficient indicates that
he YFP signal does not co-localize with MT signal at 10d)
alysis of the distribution of root mitochondria classified to their section size (i.e., the size of discrete areas display-
nd/or MT signals) using intervals of 1 mm2 (Fig. 6) showede of YFP-labelled mitochondria abundance in the range
and 1.5–2.5 mm2 sizes after 10 d of darkness (Fig. 6a). Arease of MT-marked mitochondria, especially in the range5 and 1.5–2.5 mm2, was also detected in the same roots
h and soluble sugar content and the effects of glucosentation
ermine the effect of dark treatment on carbohydrates, thestarch and soluble sugars in the roots were measured.ntent was not significantly affected by the dark treat-her at 5 or 10 d (Fig. 7a). However, the content of soluble
creased by about 50% in darkened plants after 5d of treat-g. 7b). The addition of 0.1 M glucose for 24 h producedse in the respiratory activity of roots of all treatmentso changes were observed in the abundance of either YFPy) and control plants maintained under a 10:14 h light/dark photoperiodrkness and dark gray bars represent 10d of darkness. Different letters
116 D.D. Fanello et al. / Plant Science 258 (2017) 112–121
Fig. 4. Relative amount of root proteins in plants subjected to 5 or 10 days of darkness (5d or 10d, respectively) and control plants maintained under a 10:14 h light/darkphotoperiod regime. Empty bars represent controls kept at light/dark regime, light gray bars represent 5d of darkness and dark gray bars represent 10d of darkness. Proteinswere analyzed by Cooumassie blue staining of gels (total proteins) and western blotting probed with specific antibodies against mitochondrial proteins. The data showncorresponds to the amount of protein relative to the highest value (set as 1) of each Cooumassie stained gel or western blot. Values were calculated using the ImageJ program.Sample volu ndriato Complex nd L-Gof Complex corres
or MT-la(Supplem
4. Discu
4.1. Modmitochon
A drothe progrthe quanmight acthe amounounced
labelled bof mitochof mitochO2 uptakis importall mitochfunctionsmitochonsenescenhand, sombrane po19m in
mechanoence of rmechanisstress [37
e pres [3lthous of vviab
. Theys shentriratolts inbuteince
mitobuned told bes, theity (
roo de
cultuell dld planessor le
for r me
me loaded onto each lane of the gels corresponded to 4.5 mg (FW) of roots. Mitocho IV), cyt c (Cytochome c, mitochondrial protein attached to the inner membrane) a
I). * Indicates significant differences (Student’s T-test P ≤ 0.05) compared with the
belled mitochondria between +glu and −glu treatmentsentary Fig. S3).
ssion
ifications in the quantity and activity of rootdria under continuous darkness
p in respiratory activity was observed in roots duringess of post-bolting senescence (Fig. 1). Changes in eithertity of mitochondria and/or the 19m of mitochondriacount for changes in respiration. Both the decrease innt of YFP-labelled mitochondria and the even more pro-decrease in the amount of mitochondria with 19m (i.e.,y MT) are consistent with the decrease in the contentsondrial proteins and O2 uptake. However, the abundanceondria with 19m decreased at 10d of darkness, while
e did not change between 5 and 10 days in darkness. Itant to note that Mitotracker Red probes 19m but notondrial activities, and consequently, other mitochondrial
might persist after 10 days in darkness. It was shown thatdrial integrity is conserved until the latest stages of leafce, while their number drops by 30% [36]. On the othere proteins that contribute to the modulation of the mem-
tential in response to stress could explain the decrease ofroots of senescent plants. MLS1 (MscS-Like, a family of
of thleav
Aunitis a
[38]alwaconcrespresuattri
Sthe
the arelatwourootactivwithsizedcell
ing cwoudark
Fheretheir
sensitive ion channels) leads to higher 19M in the pres-espiratory substrates but no ADP, and it may provide am for maintenance of mitochondrial redox status under]. Also, UPCs (uncopling proteins) linked to the dissipation
the initiaplant, O2but the q
l proteins: cox2 (Cytochrome c oxidase, mitochondrial protein belongingalLDH (l-Galactone-1,4-lactone dehydrogenase, mitochondrial protein
ponding control at light.
oton gradient were found down-regulated in senescing6].gh the amount of mitochondria may be indicated byolume, the quantification by unit of cross sectional area
le approach since mitochondria are discrete organelles amount of mitochondria and O2 consumption do notow a positive correlation. Under conditions of high CO2
ations, the amount of mitochondria increases but theirry activities decrease in several plant species [39]. Thesedicate that changes in O2 consumption could not be
d solely to the number of mitochondria (Figs. 9–11).the fluorescence emitted by the MT probe depends onchondrial membrane potential [30], the differences indance of mitochondria labelled by YFP and MT might be
a decrease in their metabolism, where most O2 uptake linked to uncoupled respiration. In soybean seedling
amount of AOX increases with age but alternative pathoxygen consumption in the presence of KCN) declinedt age [40] and it has been observed that AOX is synthe-novo during aging of potato tubers [41]. Also, in tobaccores the induction of AOX could prevent events trigger-eath [42]. It is possible that some alternative path activityy a role in maintaining root basal respiration in prolonged
.aves, Keech et al. [8] found similar results to those shownoots, with a decrease in the amount of mitochondria andtabolic activity becoming evident in leaves after 6 d of
tion of dark incubation. In clover roots detached from theconsumption did not change between the first 7–12 daysuantity of total protein decreased [9], which is compara-
D.D. Fanello et al. / Plant Science 258 (2017) 112–121 117
Fig. 5. Mitochondrial abundance in root cells calculated from the analysis of confocal images of plants subjected to 5 or 10 days of darkness (5d or 10d, respectively) andcontrol plants maintained under a 10:14 h light/dark photoperiod regime. a) Confocal microscopy images. Since there were no differences for control roots between 0, 5 and10d, only the 0d control is shown. Colocalization images show overlapped signals between YFP and MT (Mitotracker red) channels in white. Images show cortex cells in atangential view. Scale bars represent 25 mm. b) Number of mitochondria corresponding to YFP signal and MT (Mitotacker red) signal. Different letters indicate significantdifferences (P ≤ 0.05) according to the Kruskal-Wallis test. Normal font letters correspond to YFP differences and italics correspond to MT differences. c) Results of thecolocalization analysis as estimated through Manders coefficient. This coefficient varies between 0 (no overlap) and 1 (full overlap) where M1 (YFP → MT) corresponds tothe overlap p of MP ≤ 0.05) be
ble to thetreatmen
In maferent bupropertieand quan
frac[14].
of the YFP signal on the MT signal, and M2 (MT → YFP) corresponds to the overlatween plants subjected to 5 or 10 days of darkness and control plants.
results observed here during the 5th to the 10th d of darkt applied to the whole plant.
drialtips
ize root tips there are mitochondrial fractions of dif-oyant density with particular metabolic and structurals, such as O2 uptake, protein content, enzyme activitytity of internal membrane cristae [14]. These mitochon-
labelled m(i.e. differelated whypothes
T on the YFP signal. *Indicates significant differences (Student’s T-test
tions differ between glucose-starved and non-starved root Mitochondrial pools with or without 19m (YFP vs MT-
itochondria) or even, mitochondria with different sizesrent cross sectional area) shown in this work, could beith these different mitochondrial fractions. However, thisis deserves further studies.
118 D.D. Fanello et al. / Plant Science 258 (2017) 112–121
Fig. 6. Abu ays oa 10:14 h lig me, ligof darkness ate sig
4.2. Size
Mitocture of hdifferent
ticular sedifferent
dria are odistributeiii) they
[44]. The
in root minduced b
Mitocchondriacontributshow tha
nd oinedand pactivdue
ies, e the
t of
erentciate
of mhe otchon
ndance of root mitochondria classified in size classes for plants subjected to 5 or 10 dht/dark photoperiod regime. Empty bars represent controls kept at light/dark regi
. a) YFP-labelled mitochondria. b) MT-labelled mitochondria. Different letters indic
of root mitochondria in dark-incubated plants
hondria are pleomorphic organelles co-existing as a mix-eterogeneous morphologies in the same cell [38]. Theirsizes and morphologies seem to be regulated by a par-t of genes [43] which are differentially expressed underconditions. Three features are evident when mitochon-bserved with fluorescent probes: i) they are not equallyd in the cytoplasm; ii) they do not show similar size, and
are highly dynamic, changing their forms and positionsdata obtained here demonstrate modifications, especiallyitochondria size distribution during plant senescencey dark treatment to the whole plant.
the eobtacell
of recies
specthatmenprefassoclassOn tmito
hondria showed size heterogeneity, with smaller mito- being the more abundant and, presumably, the largestors to total root mitochondrial activity. Some evidencest the morphology and size of mitochondria change at
sets of mobservabdria labemention
f darkness (5d or 10d, respectively) and control plants maintained underht gray bars represent 5d of darkness and dark gray bars represent 10dnificant differences (Kruskal-Wallis test P ≤ 0.05).
f the cell cycle [11,13,20,43]. These evidences have been using different experimental approaches (e.g., in vitrorotoplast cultures, leaf disks, etc.) and with the additione oxygen species inducers, which may cause discrepan-
to the origin of the plant material (e.g., tissues examined,tc.) and mode of culture. However, other results suggest
size of mitochondria is not affected during the develop-leaf epidermal cells [45]. In our experiments, there is aial decrease in the amount of the smallest mitochondria,d with decreased respiration, suggesting that these are theitochondria responsible for most of respiratory activity.
her hand, it is remarkable that there are not only sets ofdria distinguished by their size but, additionally, there are
itochondria possibly with different activity. This is clearlyle by confocal microscopy, with a few or no mitochon-lled with MT after 10 d under darkness. It is important tothat the confocal observations were made on root corti-
D.D. Fanello et al. / Plant Science 258 (201
Fig. 7. Contents of starch and soluble sugars in roots of plants subjected to 5 or10 days of darkness (5d or 10d, respectively) and control plants maintained undera 10:14 hs light/dark photoperiod regime. Empty bars represent controls kept atlight/dark regime, light gray bars represent 5d of darkness and dark gray bars repre-sent 10d of darkness. a) Starch content. b) Soluble sugars content. Different lettersindicate significant differences (ANOVA P ≤ 0.05).
cal sectiothe wholmis, phlorespiratoMoreovemitochon19m pobasal acti
Both mbalance bcesses arof either
results inand a con[47,38]. Wabundan10d undeclasses rean increaallel the
not occurprocesses
4.3. The pdark trea
The eaopment ia decreastreatmening a carcontents
were anaences, busugars mConsistenwith clovtheir respdetaching
Fig. 8. Respiratory activity of roots from plants subjected to 5 or 10 days of darkness (5d or 10d, rphotoperiod regime, combined or not with the addition of 0.1 M glucose in nutrient solution. Emrepresent 5d of darkness and dark gray bars represent 10d of darkness. *Indicates significant differeand without glucose (−glu).
7) 112–121 119
ns, and respiratory activity measurements were made one root system. Therefore, other root tissues (e.g., epider-em) might explain the lack of a full agreement betweenry activity and the amount of mitochondria with 19m.r, it is interesting to note that differences between totaldria amounts (i.e., YFP-labelled) and mitochondria withint to the presence of a pool of mitochondria with onlyvity or in a “standby mode”.
itochondrial number and size depend, in part, on theetween mitochondrial fission and fusion [38]. These pro-
e frequent in plant cells [46]. For example, the disruptionof two Arabidopsis dynamin-like genes, DRP3A or DRP3B,
an increase in the size of the individual mitochondriacomitant decrease in the number of mitochondria per celle found that while smaller mitochondria were the most
t and variable (i.e., their amount decreased the most afterr darkness), the abundance of mitochondria of other sizemained fairly constant. If a fusion process were involved,se in the abundance of larger mitochondria should par-decrease in the abundance of smaller ones, which does
in our experiments. Therefore, it is probable that fusion were not involved in roots of darkened plants.
articipation of sugars in root responses to whole planttment
rly senescence of rosette leaves during post-bolting devel-n Arabidopsis plants suggests that roots may experiencee in activity due to photo-assimilate starvation. The darkt of whole plants might reproduce this phenomenon caus-bohydrate deficit in roots. To test this hypothesis, theof starch and soluble sugars in roots of dark treated plantslyzed. Since starch content didnı́t show significant differ-t soluble sugars decreased significantly, changes in solubleay be responsible for the drop of mitochondrial activity.
tly with this, Bigham and Ress [9] obtained similar resultser roots separated from the plant. These roots maintainediratory activity and starch contents for up to 30 d afterthe aboveground photosynthetic parts of the plant, while
espectively) and control plants maintained under a 10:14 h light/darkpty bars represent controls kept at light dark regime, light gray bars
nces (Student’s T-test P ≤ 0.05) between treatments with glucose (+ glu)
120 8 (201
soluble sutional enplastid ortype and
carbon flsoybean r[49] and
vation sh[10]. The
the consewhile a d
The pabated in
is partly
of roots.
ratory acaddition [root mitoof glucosenough tity of the
of MT to d19m aft
The indetachmeaddition
removal [[10] and
glucose (Soluble saside froAt the coprimarilyruled out
Becaustrates suProtein tuchlorophcarbon liness andthe mitoctional elesucrose-dcatabolisably supThereforeto root re
4.4. Conc
The dof roots
the deathdecreasesbe the codria, maiof mitochpresenceroot metareduced l
Acknowl
DDF iare resea
Nacioauthulatiniqual, Sa
endi
upplnlin
renc
.D. Noeopo988,
.B. Petab
eopp988,
. Masauficlants2010). Atketab
harkecade. Militoch
.T. van.R. Feathw.D. Vi2 (19. Keeuomiitoch
eaves.J. Binerma.P. Joepriv. Gieguring497–. Brouxcise. Zot
enescharac. Couélucosubme.C. Loeath
.M. Mrabidataboatl. A.J. Cu
mplic95–3. Yosawai
eadin. Yosawaiell de. Scotf sub. Arpaitoch
780–
D.D. Fanello et al. / Plant Science 25
gar content decreased. A. thaliana mutants lacking func-zymes related with starch metabolism, either inside the
in the cytosol, show root starch contents similar to wildvery low compared with leaves [48] suggesting a reducedux from starch to respiration. In contrast to our results,oots consume all the starch after one day under darkness
sycamore protoplast cultures subjected to sucrose depri-ow a decrease in starch content after 10 h of starvationdiscrepancies regarding changes in starch content may bequence of different experimental approaches and species,ecrease in soluble sugars is clearly evident in all cases.rtial reversion of O2 consumption decline in plants incu-
0.1 M glucose indicates that the deficit of soluble sugarsresponsible for the decrease in the respiratory activitySeveral experimental approaches show increased respi-tivity in carbohydrate starved tissues after carbohydrate10–12,14,50–52]. However, the lack of MT fluorescence inchondria from dark treated roots even after the additione might be due to the fact that 1 d incubation may not beo restore the 19m. Also, an increase of the specific activ-pool of mitochondria with 19m might explain the failureetect an increase in the abundance of mitochondria with
er glucose supply.cubation of roots for 1d in 0.1 M sucrose immediately afternt from the plant prevents the drop in respiration, but the
of sucrose is ineffective when it is provided 4 d after organ9]. Similar results were found with sycamore cell culturesmaize roots where respiration could only be restored if0.2 M) was supplied up to before 4 d of starvation [12].ugars could act in signalling [11,53] of metabolic processm acting as a source of energy for metabolic processes.ncentrations used here (0.1 M), glucose might function
as a source of energy, but a signalling function cannot be.se starch apparently is not remobilized, alternative sub-ch as proteins or amino acids may be used for respiration.rnover and degradation often occurs concomitantly with
yll and lipid breakdown, at times when plant cells aremited, for example, during extended periods of dark-
leaf senescence, and provide alternative substrates forhondrial electron transport chain [54]. In leaves a func-
ctron transfer flavoprotein (ETF) is essential for survival ofepleted cells [55,56]. This flavoprotein is involved in the
m of Leu and potentially of other amino acids, and it prob-plies mitochondria with respiratory substrates [55,56]., substrates other than soluble sugars might contributespiration under darkness.
luding remarks
ata presented here demonstrate that metabolic activityfrom naturally senescing plants is maintained beyond
of aboveground photosynthetic organs. In addition, the in respiration and amount of mitochondrial proteins maynsequence of a decrease in the abundance of mitochon-nly the small ones. The differences between the amountondria with 19m and total mitochondria suggest the
of mitochondria in a “standby state”. Part of the drop inbolic activity in plants under darkness may be due to theevels of root carbohydrates under such conditions.
dad
The
stimtechLitor
App
Sthe o013.
Refe
[1] LL1
[2] MmL1
[3] CGp(
[4] OmSA
[5] Hm
[6] JAp
[7] R3
[8] OTml
[9] Ip
[10] Ed
[11] Pd1
[12] Re
[13] Msc
[14] Igs
[15] Dd
[16] JAcN
[17] Mi2
[18] KKl
[19] KKc
[20] Io
[21] Sm1
edgements
s a post-doctoral fellow at CONICET, and CGB and JJGrchers at CONICET. This work was funded by Universi-
[22] L.D. No(1997)
[23] B.K. Nemarke(2007)
7) 112–121
nal de La Plata, Argentina (Grants 11/A258 and 11/A257).ors are grateful to Dra. María Lorenza Costa (INFIVE) forng discussions of the results and advice on biochemicales, and to Dr. Daniel González (Universidad Nacional delnta Fe, Argentina) for providing the anti-COX2 antibody.
x A. Supplementary data
ementary data associated with this article can be found, ine version, at http://dx.doi.org/10.1016/j.plantsci.2017.01.
es
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