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Research Report
Plant Cell-Cell Transport via Plasmodesmata Is Regulatedby Light
and the Circadian Clock1[OPEN]
Jacob O. Brunkard,a,b,2,3 and Patricia Zambryskia
aDepartment of Plant and Microbial Biology, University of
California, Berkeley, California 94720bPlant Gene Expression
Center, United States Department of Agriculture, Agricultural
Research Service,Albany, California 94710
ORCID IDs: 0000-0001-6407-9393 (J.O.B.); 0000-0002-2901-0320
(P.Z.).
Plasmodesmata (PD) are essential for plant development, but
little is known about their regulation. Several studies have
linkedPD transport to chloroplast-centered signaling networks, but
the physiological significance of this connection remains
unclear.Here, we show that PD transport is strongly regulated by
light and the circadian clock. Light promotes PD transport during
theday, but light is not sufficient to increase rates of PD
transport at night, suggesting a circadian gating mechanism.
Silencingexpression of the core circadian clock gene, LHY/CCA1,
allows light to strongly promote PD transport during subjective
night,confirming that the canonical plant circadian clock controls
the PD transport light response. We conclude that PD transport
isdynamically regulated during the day/night cycle. Due to the many
roles of PD in plant biology, this discovery has strongimplications
for plant development, physiology, and pathogenesis.
Plasmodesmata (PD) are nanoscopic, membrane-bound tunnels that
connect the cytosol of neighboringplant cells, transporting small
molecules, proteins aslarge as 80 kD (Kim et al., 2005; Paultre et
al., 2016),small RNAs, and viruses (Sager and Lee, 2014;Brunkard
and Zambryski, 2017). The rate of PD trans-port between cells
changes during the course of plantdevelopment (Roberts et al.,
1997, 2001; Oparka et al.,1999; Crawford and Zambryski, 2001;
Stadler et al.,2005) and in response to stress (Faulkner et al.,
2013;Caillaud et al., 2014; Cui and Lee, 2016; Lim et al.,
2016;Brunkard and Zambryski, 2017). Despite decades ofintense
research, however, very little is known aboutthe molecular
mechanisms that regulate PD transport.The only clearly established
mechanism to dynami-cally regulate PD transport is callose
deposition: undervarious stress conditions and specific
developmentalcontexts, callose (b-1,3-glucans) is synthesized
andaccumulates in the cell walls around PD, blocking trans-port
through the PD (Lee et al., 2011; Benitez-Alfonsoet al., 2013; Lim
et al., 2016). Callose deposition can be
reversed by b-1,3-glucanases, enzymes that degrade cal-lose
surrounding PD and thus restore transport throughPD (Zavaliev et
al., 2011).To identify molecular pathways that regulate PD
transport, we and others have conducted forward ge-netic
screens. These screens to identify factors control-ling transport
through PD have repeatedly revealedthat chloroplasts influence PD
transport (Provencheret al., 2001; Benitez-Alfonso et al., 2009;
Burch-Smithand Zambryski, 2010, 2012; Burch-Smith et al.,
2011;Stonebloom et al., 2012; Brunkard et al., 2013; Carlottoet
al., 2016; Bobik et al., 2019). This has led to a para-digm shift,
focusing less on the role of structuralchanges directly at PD and
more on how cellularphysiology influences the function of PD.
Althoughmany groups have now demonstrated that chloroplastfunction
and PD transport are tightly connected, thebiological significance
of this relationship betweenchloroplasts and PD remains
unresolved.Given the connection between PD transport and
chloroplast physiology, we hypothesized that PDtransport might
be sensitive to light. A pioneeringstudy in maize (Zea mays)
seedlings demonstratedthat PD transport decreases during
deetiolation, whendark-grown seedlings are first exposed to light
andchloroplast biogenesis is initiated (Epel and Erlanger,1991). PD
transport also decreases at the midtorpedostage of Arabidopsis
(Arabidopsis thaliana) embryogen-esis, which is when embryos
initiate chloroplast bio-genesis. PD transport does not decrease at
this stage ofembryogenesis in mutants defective in chloroplast
bi-ogenesis, including ise1, ise2, clpr2, and uL15c (Burch-Smith et
al., 2011; Carlotto et al., 2016; Bobik et al.,2019). Therefore, at
least in some developmental andphysiological contexts, changes in
PD transport arecoordinated with chloroplast biogenesis.
1This work was supported by the National Institutes of
Health(grant 5-DP5-OD023072 and a graduate research fellowshipto
J.O.B.).
2Author for contact: [email protected] author.The
author responsible for distribution of materials integral to
the
findings presented in this article in accordance with the policy
de-scribed in the Instructions for Authors (www.plantphysiol.org)
is:Jacob O. Brunkard ([email protected]).
J.O.B. conducted experiments and wrote the original article;
J.O.B.and P.Z. designed experiments, supervised the project, and
edited thearticle.
[OPEN]Articles can be viewed without a
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Plant Physiology�, December 2019, Vol. 181, pp. 1459–1467,
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Beyond these studies indicating that light-dependentchloroplast
biogenesis influences PD transport, little isknown about how light
influences plant cell-celltransport. One report suggested that PD
transport canincrease when starvation is induced by detaching a
leafand transferring it to complete darkness (Liarzi andEpel,
2005), but whether PD transport was impactedby light signaling or
starvation stresses in this experi-mental system was not resolved.
An ultrastructuralstudy of several C4 grass species found that PD
fre-quency in the cell wall increases when seedlings aregrown under
higher light intensities (Sowi�nski et al.,2007). Here, we combine
genetic and physiologicalapproaches to show that PD transport is
dynamicallyregulated by light and the circadian clock throughoutthe
diurnal cycle.
RESULTS
PD Transport Rates Are Higher during the Day
As a preliminary experiment, we performed a simplequalitative
assay to monitor PD transport in Arabi-dopsis during the day or
night using fluorescent tracers.Arabidopsis seedlings were
germinated on plates con-taining Murashige and Skoog medium and
grown ina 12-h-light/12-h-dark light cycle. Three hours afterdawn
or dusk, a fluorescent tracer was applied toseedlings by cutting
the plant just below the base ofthe shoot and applying a small
volume (;10 mL) offluorescent tracer to the remaining root
portion(Supplemental Fig. S1A). Seedlings were kept onplates at
high relative humidity throughout the exper-iment to limit
transpiration and 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS)
transport via xylem vessels.After 10 min, the cotyledons of
seedlings were visual-ized. Low-molecular-mass (;500 D) HPTS (also
knownas pyranine) moved rapidly and extensively throughthe ground
tissue via PD from the cut surface of the rootinto the hypocotyl
and then into the cotyledons dur-ing the day (Supplemental Fig.
S1B) but not at night(Supplemental Fig. S1C). This result implied
that PDtransport is higher during the day than at night.
To better quantify the differences in PD transportbetween the
day and the night, we used a GFP move-ment assay in Nicotiana
benthamiana leaves. Plants weregrown in a 12-h-light/12-h-dark
light cycle under oth-erwise constant conditions. Agrobacterium
tumefacienscarrying 35SPRO:GFP T-DNAwas infiltrated into leaveswith
very low inoculum (OD600nm 5 1024, less than 100cells total
infiltrated per leaf) at either dusk or dawn,after which GFP
movement from distinct, individualtransformed cells in the leaves
was visualized 36 to 60 hpost infiltration. GFP expression
typically becomesstable;24 h after infiltration (sufficient time
for T-DNAtransfer and transcription) and remains stable for 72 to96
h, at which time host RNA-silencing mechanismsbegin to reduce
transgene expression (Voinnet et al.,2015). The Cauliflower mosaic
virus 35S promoter is
widely used for strong and consistent gene expressionand is not
regulated by diurnal cues or the circadianclock (Suárez-López et
al., 2001). Across all experiments(except for the photoperiod
experiment, described inmore detail below and in "Materials
andMethods"), thefourth expanded leaf from the top of a 5-week-old
plantwas agroinfiltrated. For consistency, only similar plantsand
leaves of nearly identical size and agewere used forthese assays,
and GFP movement was only visualizedin the proximal 25% of the
agroinfiltrated leaf. Eachtransformed cell is reported as an
independent sample(n), the experiments were conducted using at
least eightto ten separate plants, and each experiment was
re-peated at least three times to ensure reproducibility.Sampling
details are described in “Materials andMethods” and in Supplemental
Table S1. Results of thismovement assay are presented as the number
of cellsGFP has spread to from the transformed cell (examplesin
Fig. 1B). Compared with most other approaches, thismethod is
minimally invasive, since N. benthamianalacks the
Brassicaceae-specific A. tumefaciens immunityreceptor EF-Tu
RECEPTOR (EFR), leaves are infiltratedwith only a few dozenA.
tumefaciens cells (which are faroutnumbered by endogenous
phyllobacteria), theagroinfiltration does not require significant
pressure orwounding in N. benthamiana leaves, and no
foreignmolecules are injected into cells by pressure orwounding;
instead, individual transformed leaf cellsproduce the fluorescent
tracer GFP, permitting nonin-vasive imaging. Moreover, unlike many
fluorescentdyes, which can be sequestered in vacuoles or exportedto
the apoplast, GFP remains symplastic, freely movingthrough cytosol
and nucleosol but not trafficking acrossmembranes, and thus only
moves between cells via PD(Crawford and Zambryski, 2001).
Whether leaves were infiltrated at dawn or dusk,GFPmoved the
same distance after 48 h (to 336 1 cells,n $ 106 transformed cells,
P 5 0.26; Fig. 1C), demon-strating that the time of infiltration
had no impact onthe observed rates of PD transport. When
observedafter 36 h, however, GFP moved significantly more inleaves
that have experienced two days and one nightthan in leaves that
have experienced one day and twonights (to 256 1 cells [dawn
infiltration] versus to 1761 cells [dusk infiltration], n$ 117
transformed cells, P,1027; Fig. 1C). Similarly, when observed after
60 h, GFPalso moved significantly more in leaves that have
ex-perienced three days and two nights than in leaves thathave
experienced two days and three nights (to 54 61 cells [dawn
infiltration] versus to 35 6 1 cells [duskinfiltration], n $ 102
transformed cells, P , 10210;Fig. 1C). Thus, GFP moves more in
plants that haveexperienced two days (daytime light periods) than
inleaves that have experienced one day, and GFP movesthe most in
leaves that have experienced three days,demonstrating that the rate
of PD transport is higherduring the day than at night.
Remarkably, an additional night has little impact onGFP
movement, suggesting that PD transport at nightis relatively
limited. GFP movement is only slightly
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lower in leaves 36 h after infiltration at dawn (i.e. leavesthat
experienced two days and one night) than in leaves48 h after
infiltration (i.e. leaves that experienced twodays and two nights;
GFP moved to 25 6 1 cells 36 hafter infiltration at dawn versus to
336 1 cells 48 h afterinfiltration, n$ 106 transformed cells, P,
1023; Fig. 1C,bottom). PD transport was not significantly different
inleaves observed 60 h after infiltration at dusk (i.e. leavesthat
experienced two days and three nights) than inleaves observed 48 h
after infiltration at dusk (60 h: GFPmoved to 35 6 1 cells versus
48 h: GFP moved to 33 61 cells, n $ 102 transformed cells, P 5 0.2;
Fig. 1C,bottom) or 48 h after infiltration at dawn (n $
111transformed cells, P 5 0.91; Fig. 1C, bottom). Theseresults
support our preliminary hypothesis that PDtransport is, in fact,
dramatically lower at night thanduring the day.
Diurnal Changes in PD Transport Are Not Due toCallose
Deposition
PD transport can be restricted during stress re-sponses and
cellular differentiation by deposition of apolysaccharide, namely
callose, in the plant cell wallsurrounding PD. We therefore
investigated whethercallose levels around PD are higher at night
than duringthe day, which could explain the decreased rate of
PDtransport at night. We infiltrated leaves with Aniline
Blue using state-of-the-art methods (Cui and Lee, 2016)to stain
callose in leaves 3 h after dawn or 3 h after duskand then used
confocal laser-scanning fluorescencemicroscopy followed by
quantitative image analysis tomeasure callose levels at PD in leaf
cell walls. For theseexperiments, each sample is the average
fluorescenceintensity of Aniline Blue at PD in four separate fields
ofview in a leaf, with comparable numbers of PD in-cluded in each
field of view, such that the relative cal-lose levels are based on
measurements of fluorescencefrom hundreds of individual PD. Callose
levels at PDare somewhat lower at night than during the day (1
60.25 arbitrary units of fluorescence, day; 0.86 6 0.2 ar-bitrary
units of fluorescence, night; n 5 8 plants, P 50.08; Fig. 2).
Therefore, the difference in PD transport islikely not due to a
reversible, nocturnal deposition ofcallose at PD.Another simple
explanation for the change in PD
transport could be reduced ATP availability at night,for
example, if PD transport is largely driven by cyto-solic convection
powered by ATP-dependent cyto-skeletal activity (Pickard, 2003).
Inhibition ofchloroplast and/or mitochondrial activity (and
thusinhibition of ATP generation), however, can cause ei-ther
increased or decreased transport (Benitez-Alfonsoet al., 2009;
Stonebloom et al., 2012), suggesting that thissimple model is not a
sufficient explanation. Here, us-ing virus-induced gene silencing
(VIGS), we testedwhether silencing the expression of AtpC,
which
Figure 1. PD transport is higher during the day than at night.
A, To measure rates of PD transport, we used a quantitative
GFPmovement assay. A very low inoculum of A. tumefaciens cells
(less than 100 bacterial cells) was gently infiltrated by syringe
intoN. benthamiana leaves so that a handful of individual epidermal
cellswere transformed to expressmonomericGFP. GFP spread
toneighboring epidermal cells was then quantified 48 h after
infiltration (or as indicated in each experiment). Illustrated
examples ofGFPmovement are shown here. Movement is scored by
counting the number of neighboring cells to which GFP has moved
(darkgreen) from the transformed cell (bright green). B,
Representative confocal microscopy images of the GFP movement
assay. GFPfluorescence is brightest in the nuclei in cells
neighboring the transformed cell. Bar 5 100 mm. C, N. benthamiana
leaves wereagroinfiltrated to express GFP at either dusk (top) or
dawn (bottom) in 5-week-old plants grown under 12-h-light
(yellow)/12-h-dark (blue) cycles. GFP movement from the transformed
cell was then assayed 36, 48, or 60 h later. In leaves infiltrated
at dusk(top), GFP movement significantly increased during the
second day but did not change during the third night. In leaves
infiltratedat dawn (bottom), GFP movement somewhat increased during
the second night but dramatically increased during the third day.*,
P , 1023 and **, P , 1025; n.s., not significant.
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encodes a subunit of the chloroplast ATP synthase, in-fluences
PD transport. We chose this gene because si-lencing AtpC has been
shown to promote theintercellular spread of Tobacco mosaic virus
(Bhat et al.,2013). We observed increased PD transport after
si-lencing AtpC, from 28 6 2 cells in mock (TRV::GUS)plants to 47 6
2 cells in TRV::AtpC-knockdown leaves(n $ 21 transformed cells, P ,
0.003; Fig. 2, C and D).These results support the hypothesis that
higher ATPlevels do not correlate with increased PD transport
andhint that increased spread of Tobacco mosaic virus
inTRV::AtpC-knockdown plants could be a consequenceof increased PD
transport.
PD Transport Is Regulated by the Plant Circadian Clock
High rates of PD transport during the day could becaused
directly by light signaling or could be mediatedby light-entrained
signaling pathways controlled by thecircadian clock (Harmer, 2009).
To distinguish betweenthese possibilities, we first tested whether
transferringN. benthamiana plants grown in
16-h-light/8-h-darkcycles to either constant light or constant dark
impactedPD transport. We conducted these first experimentsusing
16-h-light/8-h-dark cycles (Fig. 3A) and thenalso tested
12-h-light/12-h-dark cycles (describedbelow; Fig. 3, B and C), with
comparable results. After48 h, there was no significant difference
in GFPmovement between plants under constant light orunder
continued day/night cycles (16-h-light/8-h-dark cycle), with GFP
moving 35 6 3 cells underconstant light versus 336 3 cells under
cycling light/dark conditions (n $ 60 transformed cells, P 5
0.35;Fig. 3A). GFP movement was severely lower in plantstransferred
to constant dark, however, moving anaverage of only 106 1 cells (n$
60 transformed cells,P , 10223; Fig. 3A). Light is therefore
necessary forthe higher rate of PD transport during the day,
butconstant light is insufficient to significantly promotePD
transport.
In plants, the circadian clock serves primarily to an-ticipate
regular environmental changes and to gate re-sponses to irregular
or fluctuating stimuli (Harmer,2009). For example, the circadian
clock prevents theinduction of photosynthesis-associated nuclear
genesby brief bursts of light at night, preventing wastefulprotein
synthesis (Kay and Millar, 1992; Andersonet al., 1997). Gating can
be observed by entrainingplants under regular day/night cycles,
moving theminto constant conditions, and then applying a
stimulus(e.g. light) at either subjective day or subjective
night;ungated responses will be observed in both cases, butgated
responses will differ depending on when thestimulus is applied. We
next tested whether a stimulusgating mechanism at night could
explain the insuffi-ciency of constant light to increase the rates
of PDtransport described above (Fig. 3A). N. benthamianaplants were
grown under 12-h-light/12-h-dark photo-periods and then transferred
to constant darkness for 72h. One group of plants was then exposed
to 12 h of lightonly during the second subjective day (1L during
day),whereas a second group of plants was exposed to 12 hof light
only during the second subjective night (1Lduring night), and a
third group of plants was main-tained in constant darkness (no
light). As expected, PDtransport was low in constant darkness (21 6
4 cells).PD transport only slightly increased when 12 h of lightwas
applied during the second subjective night (to 2163 cells, n$ 59
transformed cells, P5 0.03) butwasmuchhigher when light was applied
during the second sub-jective day (to 326 4 cells, n$ 70
transformed cells, P,1025; Fig. 3, B and C).
To confirmwhether the circadian clock is responsiblefor gating
PD transport at night, we took a genetic ap-proach and used VIGS to
knock down the expression ofthe core circadian clock gene, LATE
ELONGATEDHYPOCOTYL (LHY). Reverse transcription quantita-tive PCR
(RT-qPCR) analysis confirmed that VIGS re-duced LHY expression to
2.8% of mock-infected LHYtranscript levels in leaves collected 1 h
after subjectivedawn, whenwild-type LHY expression peaks. LHY
and
Figure 2. A and B, PD callose deposition is not significantly
different between day and night. Fluorescence intensity of
AnilineBlue-stained leaves was assayed using confocal scanning
laser microscopy in the leaf epidermis ofN. benthamiana collected 3
hafter dawn or 3 h after dusk. Callose levels were slightly lower
at night than during the day (n5 8 leaves, P5 0.08). a.u.,
Arbitraryunits; ns, not significant. Bar5 100 mm. C, Reducing
ATPavailability by silencing AtpCwith VIGS significantly increased
the rateof GFP transport (n$ 21 transformed cells; *, P5 0.003),
demonstrating that light-dependent chloroplast ATP synthesis and
PDtransport do not positively correlate. D, Representative images
of the GFP movement assay in mock (TRV::GUS) and atpC(TRV::AtpC)
plants. Bar 5 100 mm.
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its recently evolved paralog, CCA1, are required tomaintain
circadian rhythms in Arabidopsis; similarly,silencing LHY (there
are no distinct CCA1 orthologs inSolanaceae) abolishes circadian
rhythms in Nicotianaspp. (Yon et al., 2016). We conducted this
experimentside by side with the gating experiment describedabove,
which involved mock infection with the VIGSvectors. As expected, PD
transport was as low in con-stant darkness in TRV::LHY-knockdown
leaves as inmock leaves (176 1 cells in mock versus 166 1 cells
inTRV::LHY, n$ 70 transformed cells, P5 0.75; Fig. 3C).Unlike the
mock treatment leaves, however, TRV::LHY-knockdown leaves
significantly increased PD transportwhether light was applied
during the day or the night(to 506 2 cells when applied during the
subjective day,n $ 31 transformed cells, P , 1027, or to 40 6 1
cellswhen applied during the subjective night, n $ 71transformed
cells, P , 1029; Fig. 3C). Thus, gating bythe LHY-dependent
circadian clock is required to pre-vent light from inducing higher
rates of PD transport atnight. These results demonstrate that PD
transport isunder tight regulation by both light and the
circadianclock to promote intercellular trafficking during the
dayand limit movement between cells at night.
Diurnal PD Transport Rates Decrease with Leaf Age ButAre Not
Correlated with Photoperiod
We next tested whether PD transport is lower inplants grown with
shorter daylengths, since the aboveresults show that PD transport
is positively regulatedby light. Daylength can impact the rate of
leaf emer-gence, however, such that it is difficult to know a
prioriwhich leaf stage(s) to compare. Instead of selecting asingle
leaf stage, therefore, we assayed PD transport inseveral leaves of
mature plants grown under differentphotoperiods. We grew N.
benthamiana plants under12- or 16-h daylengths, recording the dates
of leafemergence throughout their development. Six weeksafter
germination, we simultaneously assayed PDtransport with the GFP
movement assay in all leavesbetween 10 and 30 d old. This
experiment was repli-cated three times, and the dates of leaf
emergence didnot change across replicates (but were different
be-tween plants grown under 12- or 16-h daylengths, asshown; Fig.
4). PD transport, as measured by thequantitative GFP movement
assay, decreases in anapproximately linear pattern with respect to
leaf age,regardless of photoperiod conditions (Fig. 4).
Remark-ably, there is no significant difference in the rate of
PD
Figure 3. PD transport is regulated by the circadian clock.
Light treat-ment is represented by yellow, dark and treatment is
represented byblue. Line i of both A and B shows the day/night
cycles that would beexperienced by plants if they were not
transferred to new light regimes.A, After growing in
16-h-light/8-h-dark cycles, N. benthamiana leaveswere
agroinfiltrated at dawn or dusk (as indicated by red
infiltrationarrows) to express GFP. GFP movement was then assayed
after 48 h (asindicated by green observation arrows) of continued
light/dark cycles(lines ii and iii), or 48 h of constant light
starting at the end of the day(line iv), or constant darkness
starting at the end of the night (line v).Constant light did not
affect PD transport, but constant darkness sig-nificantly decreased
PD transport (**, P, 10223; ns, not significant). B,PD transport
was assayed in mock-treated plants (TRV::GUS) that hadbeen growing
in 12-h-light/12-h-dark cycles and then transferred toconstant dark
conditions; GFP movement was assayed 72 h afteragroinfiltration.
Subjective days and nights are shown in line i. Treat-ment with 12
h of light during the second subjective day after agro-infiltration
(line iii) strongly increased PD transport compared with
PDtransport in constant dark (line ii), but treatmentwith 12 h of
light duringthe second subjective night (line iv) had no
significant effect on PDtransport. C, Leaves of mock-treated plants
(left) or TRV::LHY knock-downs (right) under 12-h-light/12-h-dark
photoperiod conditions wereagroinfiltrated to express GFP at
subjective dawn. Plants were trans-ferred to the dark and either
maintained in complete darkness for 72 h(dark gray bars) or exposed
to 12 h of light during the second subjective
night (light gray bars) or 12 h of light during the second
subjective day(white bars). Mock-treated plants distinguished
between light appliedduring subjective night or subjective day,
significantly increasing PDtransport only after exposure to light
during the day. TRV::LHY knock-downs did not distinguish between
subjective night or subjective day,significantly increasing PD
transport after exposure to light during eithertime period. **, P ,
1027; ns, not significant.
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transport of plants grown under 12- or 16-h photope-riods, as
tested by an analysis of covariance (ANCOVA)with respect to leaf
age (n $ 59 cells, ANCOVA P 50.27, homogeneity of regressions P 5
0.25). Thus, therate of PD transport is indistinguishable between
plantsthat have developed under either 12- or 16-h photope-riods
when comparing leaves of the same age.
DISCUSSION
Here, we have shown that the rate of moleculartransport through
PD changes during the diurnal cycle.GFP and fluorescent tracers
move more rapidly be-tween plant cells during the day than at
night. Thehigher rates of PD transport during the day are
lightdependent. Light is not sufficient to increase the rate ofPD
transport at night, however, because of regulationby the circadian
clock. Thus, multiple regulatory
mechanisms dynamically control PD transport duringthe course of
the diurnal cycle.
A previous report argued that light down-regulatesPD transport
in leaves (Liarzi and Epel, 2005) ratherthan promoting PD
transport, as we report here. Theseearlier studies were based on
very different physio-logical conditions, however. One study showed
that PDtransport is higher in young sink leaves after they
aredetached from plants grown under 16-h-light/8-h-darkphotoperiods
and transferred to nutrient-free media inconstant darkness (Liarzi
and Epel, 2005). Transferringindividual leaves to constant
darkness, however, in-duces starvation responses, whereas
transferring entireplants to constant darkness does not (Weaver
andAmasino, 2001). Thus, we assert that the opposite re-sults
obtained in the previous report are likely due tocomplications from
detaching the leaf, whereas, in ourexperiments, all conditions were
kept constant exceptfor the light environment.
Our results measuring PD transport in leaves ofmultiple ages on
the same plant demonstrate that PDtransport within the leaf
epidermis gradually changesas leaves age, rather than abruptly
shifting from highPD transport to low PD transport during the
sink-to-source transition. As the plant shoot develops,
leavestransition from heterotrophic sinks for
carbohydrates,nitrogen, and other resources to
photoautotrophicsources of carbohydrates for the rest of the
plant(Masclaux et al., 2000). The sink-to-source
physiologicaltransition is supported by the restriction of PD
trans-port in source leaves, which can then actively loadsugars
into the phloem via directional sugar trans-porters at the plasma
membrane without allowing thesugars to move back into the leaf
through PD (Turgeon,1989). In this way, restriction of PD transport
permitsthe formation of a steep carbohydrate concentrationgradient
in the phloem, shifting the water potential topromote rapid
transport of molecules through thephloem and driving vascular plant
growth and devel-opment. Previous studies often used qualitative
mea-surements to define the sink-to-source transition in
PDtransport (specifically, fluorescent constructs that eitherdo or
do not move from the phloem into the groundtissue [Roberts et al.,
1997; Imlau et al., 1999]) and em-phasized a bimodal sink-to-source
transition withinleaves: PD transport is restricted first in the
distal regionof the leaves, and then this restrictionmoves
proximallyuntil PD transport is low throughout the entire
leaf(Oparka et al., 1999). The data presented here demon-strate
instead that PD transport decreases graduallyand quantitatively as
a leaf ages.
Although PD transport rates are higher during theday, we
observed no significant effect of daylength onPD transport in
plants grown under 12- or 16-h pho-toperiods. One hypothesis to
address this result is thatPD transport primarily occurs during
only the earlyhours of the day, not the late afternoon. This is
sup-ported by our results comparing mock (TRV::GUS)versus TRV::LHY
plants (Fig. 3C): when plants weretransferred to darkness for 72 h
but treated with 12 h of
Figure 4. A, Leaf emergence was recorded every day for N.
ben-thamiana plants grown under 12- or 16-h daylengths. The age of
eachleaf (in number of days) in the mature plant at the time of the
GFPmovement assay (6 weeks after germination) is shown. Cotyledons
arenot included in this diagram because they senesced within the
first6 weeks of growth. B, AverageGFPmovement in leaves of
different agesis shown for plants grown with 12-h daylengths
(purple boxes) or 16-hdaylengths (orange boxes). GFP movement was
assayed 48 h afteragroinfiltration with a low inoculum of A.
tumefaciens that transformedcells to express GFP. PD transport
declines in the same linear rela-tionship with leaf age in both
sets of plants (n $ 59 transformed cells,ANCOVA P 5 0.27,
homogeneity of regressions P 5 0.25). The linearrelationship is
depicted with a gray dashed line with slope 23.2 and yintercept
92.
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light during the subjective day, GFP movement wasgreater in
TRV::LHY knockdowns (506 4 cells) than inmock-treated plants (33 6
3 cells). The most straight-forward explanation for this difference
is that the cir-cadian clock gates PD transport during a portion of
theday. Indeed, many light-dependent diurnal processesare gated by
the circadian clock during the day. Forexample, photosynthetic
rates are typically highestduring the morning and significantly
lower in the lateafternoon, when the benefits of
photosynthesizingmore sugars are outweighed by the costs
associatedwith photosynthesis (e.g. water loss via stomata
exac-erbated by higher temperatures; Parry et al., 1993). It
ispossible that PD transport rates, like photosyntheticrates, are
also higher during the first hours of the dayand then decrease
under regulation by the circadianclock. This hypothesis would also
explain why the re-duction of daylength from a 16-h to a 12-h
photoperiodwould not strongly impact PD transport, because
theduration of daylight at the end of the day is reducedand most PD
transport would primarily occur duringthe first few hours after
dawn.
CONCLUSION
As a working model, we propose that light promotesPD transport
during the day and that darkness and thecircadian clock repress PD
transport at night and pos-sibly also during part of the day. This
regulation islikely independent of the well-established callose
de-position mechanism that can restrict PD transport. Thepotential
physiological implications of this work areclear: cell-cell
transport and signaling is strongly mod-ulated by the time of day.
Ongoing efforts to dissect thebiology controlled by PD transport in
plants, includinghormone transport and regulatory networks
(Benitez-Alfonso et al., 2013; Besnard et al., 2014; Han et al.,
2014;Lim et al., 2016), host-pathogen interactions (Khanget al.,
2010; Wang et al., 2013; Caillaud et al., 2014),and protein and
small RNA trafficking (Gallagher et al.,2014; Brunkard and
Zambryski, 2017), will be informedby the discovery that PD
transport is tightly and dy-namically regulated by the circadian
clock.
MATERIALS AND METHODS
Plant Growth Conditions
Nicotiana benthamiana accession Nb-1 plants were grown as
described(Brunkard et al., 2015) at 22°C to 24°C on light carts
under ;100 mE m22 s21
photosynthetically active radiation using Sylvania Gro-Lux Wide
Spectrumbulbs with either 12- or 16-h daylengths, as indicated in
the text. Plants weretransferred to complete darkness but otherwise
kept in constant conditions,where noted in the text. For the
photoperiod experiment, plants were photo-graphed every day after
germination in order to accurately record leaf ages.
Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 plants
were grown at22°C to 24°C in growth chambers under ;100 mE m22 s21
photosyntheticallyactive radiation using Sylvania Gro-Lux Wide
Spectrum bulbs with 12-h day-lengths. Seeds were surface
sterilized, stratified in the dark at 4°C for 2 d, andsown on 0.53
Murashige and Skoog medium (Caisson Labs) with 0.8% (w/v)
agar and pH adjusted to 5.5. Seedlings were kept on plates to
maintain highrelative humidity throughout all experiments.
Agroinfiltrations
Agrobacterium tumefaciens strain GV3101 was grown overnight in
lysogenybroth medium at 28°C, 250 rpm, with kanamycin, gentamicin,
and rifampicin(each at 50mgmL21). Cultureswere resuspended in
infiltrationmedium (10mMMgCl2, 10 mM MES, and 200 mM
acetosyringone, pH 5.6, adjusted with KOH) toOD600nm 5 1024 for GFP
movement assays or OD600nm 5 1 for VIGS, as pre-viously described
(Brunkard et al., 2015).
Assaying PD Transport with GFP Transformation inSingle
Leaves
Except for the photoperiod experiment (described in detail in
the "AssayingPD Transport with GFP Transformation in Mulitple
Leaves" section), allmovement assayswere conducted using the fourth
expanded leaf of 5-week-oldplants. GFP movement assays were
conducted as previously described(Brunkard et al., 2015; Fig. 1),
observing GFP movement in only the proximal25% of the leaf. For
each experiment, two, three, or four plants were assayed foreach
condition per replicate (observing GFP movement from up to 30
indi-vidual transformed cells selected randomly from one leaf on
each plant), andthe entire experiment was replicated three times,
so that each experiment wasconducted in at least eight to 10
plants. Movement is scored by counting thenumber of neighboring
cells to which GFP has moved from the original,transformed
cell.
Assaying PD Transport with GFP Transformation inMultiple
Leaves
For the photoperiod experiment, GFP movement assays were
performed aspreviously described (Brunkard et al., 2015), with the
difference that A. tume-faciens carrying the 35SPRO:GFP T-DNA
binary vector was infiltrated into everyleaf between 10 and 30 d
old of the plant in 6-week-old plants. GFP movementwas observed in
all leaves in the proximal 25% region of the leaf using
threebiological replicates. Leaves beyond 30 d old were not
included because GFPwas generally unable to move beyond the
transformed cell, and thus the GFPmovement assay was not
sufficiently sensitive to assay changes in PD transportin these
leaves. Leaves less than 10 d old were not included because GFP
spreadso far (to as many as 100 cells) that it became impractical
to consistently andaccurately distinguish spreading GFP foci.
Assaying PD Transport with Fluorescent Tracers
HPTS (Sigma-Aldrich)was dissolved in 0.53Murashige and
Skoogmedium(Caisson Labs) at 1 mg mL21. Ten microliters of HPTS was
applied to theproximal root remaining immediately after cutting off
the distal root near theroot-shoot connection in 4-d-old
Arabidopsis seedlings. Seedlings were kept onplates throughout the
experiment to maintain high relative humidity, which wefound
prevented HPTS from traveling apoplastically (via xylem vessels).
Cot-yledons were then observed approximately 10 min after
application of the dye.At least three individual seedlings were
assayed per experiment, and the ex-periment was repeated three
times, with representative images shown.
VIGS
Silencing triggers were cloned as previously described (Brunkard
et al.,2015). Briefly, RNA was isolated from N. benthamiana Nb-1
with the Spec-trum Plant Total RNA kit (Sigma-Aldrich) with
on-column DNase I digestion(New England Biolabs). cDNA was
synthesized from RNA using randomhexamers and SuperScript III
reverse transcriptase (Fisher Scientific). Silencingtriggers were
amplified with Phusion DNA polymerase (New England Biol-abs),
digested with XbaI and XhoI (New England Biolabs) alongside
digestionsof pYL156 (Liu et al., 2002), and ligated with Promega T4
DNA ligase (FisherScientific). Ligations were transformed into
XL1-Blue Escherichia coli, mini-prepped (Bioneer), and Sanger
sequenced to confirm insertion sequences. TheAtpC trigger was
cloned using the same sequence as previously reported (Bhatet al.,
2013), with oligonucleotides 59-gactctagaTTCCTAACCATAACTCATCAGG-39
and 59-gatctcgagAAAACATCATCAGCAATGG-39 (XbaI and XhoI
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restriction sites were introduced by PCR with these
oligonucleotides and areindicated in lowercase).
Two young leaves were infiltrated with equal inocula of A.
tumefacienscarrying binary vectors encoding the two Tobacco rattle
virus VIGS constructs(TRV1 and TRV2-trigger; Liu et al., 2002). A
TRV2-GUS trigger was used as anegative control, because GUS does
not have any sequence similarity to en-dogenous transcripts in N.
benthamiana (Stonebloom et al., 2009). A TRV2-NbPDS (PHYTOENE
DESATURASE) trigger was used as a positive controlfor silencing,
because pds knockdowns exhibit strong photobleaching pheno-types
that can be monitored visually (Stonebloom et al., 2009). Silencing
effi-ciency RT-qPCR analysis and GFP movement assays were conducted
14 d afterinfiltration.
RT-qPCR
N. benthamiana upper leaves, comparable to the leaves used for
the GFPmovement assay, were collected 2weeks post infectionwith
either TRV::LHY orTRV::GUS, a mock control (Stonebloom et al.,
2009). Tissue from three replicateplants was collected 1 h after
dawn, when LHY is strongly expressed in wild-type plants. RNA was
isolated with the Spectrum Plant Total RNA (Sigma-Aldrich) kit with
on-column DNase I digestion (New England Biolabs). cDNAwas
synthesized from RNA using oligo(dT)18 and SuperScript III
reversetranscriptase (Fisher Scientific). qPCR was performed in
parallel for all samplesusing a validated reference gene (EF1a) for
N. benthamiana VIGS experiments(Liu et al., 2012) and primers
specific to LHY: 59-TAGCTGGAGATGCTGGGAAT-39 and
59-TGAAAAGAGCCTGGAATGCT-39.
Assaying PD Callose Deposition with Aniline Blue
Leaveswere infiltratedwith sterile 0.01%(w/v)AnilineBlue
(Sigma-Aldrich)in 10 mM K3PO4 (pH 12; Zavaliev and Epel, 2015) and
left to stain for 1 h in thedark (Cui and Lee, 2016). Leaves were
then mounted on slides, and the abaxialepidermis was imaged with a
Zeiss 710 confocal scanning laser microscopeequippedwith aW
Plan-Apochromat 40x/1.0 DICM27 objective. Four fields ofview from
the proximal 25% of the leaf were imaged from each leaf. Each PDwas
identified manually in ImageJ, and Aniline Blue fluorescence
intensity perPDwas recorded. Average callose levels per PD from
each leaf were consideredindependent samples. Callose levels per PD
were then averaged across allleaves sampled. One leaf from four
plants was assayed for each condition perexperiment, and the
experiment was repeated twice.
Microscopy
GFPwas observed in the epidermis ofN. benthamiana leaves using
aZeiss 710confocal scanning laser microscope. Identical settings
(such as laser strengthand gain) were used for all GFP experiments.
HPTS and Aniline Blue stainingwere also observed with a Zeiss 710
confocal scanning laser microscope. Forquantification, Aniline Blue
staining was observed with a W Plan-Apochromat40x/1.0 DIC M27
objective, and PD fluorescence intensity in images was ana-lyzed
using ImageJ. FITC-Dextran unloading in roots was observed with
epi-fluorescence illumination using a Zeiss AxioImager M2.
Quantification and Statistical Analysis
Each cell transformed to expressGFPwas considered an independent
samplefor the movement assay. Movement assay results are presented
as the averagemovement of GFP and SE. Differences in GFP movement
between two givenconditions were compared using unpaired
heteroscedastic Student’s t tests inExcel, with P , 0.01 considered
significantly different.
Independent samples of callose levels (as described above) were
comparedusing unpaired heteroscedastic Student’s t tests in Excel,
with P , 0.01 con-sidered significantly different.
AnANCOVA (Fig. 4B)was conducted using Excel, with P, 0.01
consideredsignificantly different.
Accession Numbers
The sequence of NbLHY can be found in the SolGenomics data
libraries(https://solgenomics.net) under accession number
Niben101Scf02026g01002.1.
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. PD transport is higher during the day
thanat night.
Supplemental Table S1. Detailed sampling information for GFP
move-ment assays.
ACKNOWLEDGMENTS
We thank Steven Ruzin and Denise Schichnes at the College of
NaturalResources Biological Imaging Facility (University of
California, Berkeley) andDe Wood and Tina Williams at the U.S.
Department of Agriculture WesternRegional Research Center for
microscopy support. We thank Anne M. Runkel(University of
California, Berkeley) for generously providing assistance
withconducting experiments. We thank Claire Bendix (University of
California,Berkeley) for constructive comments on experimental
design and the article.
Received April 16, 2019; accepted September 24, 2019; published
October 10,2019.
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