plant cell-cell transport viaplasmodesmata is regulated...plant cell-cell transport viaplasmodesmata...

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 subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.19.00460

Plant Physiology�, December 2019, Vol. 181, pp. 1459–1467, www.plantphysiol.org � 2019 American Society of Plant Biologists. All Rights Reserved. 1459

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http://orcid.org/0000-0001-6407-9393http://orcid.org/0000-0001-6407-9393http://orcid.org/0000-0002-2901-0320http://orcid.org/0000-0002-2901-0320http://orcid.org/0000-0001-6407-9393http://orcid.org/0000-0002-2901-0320http://crossmark.crossref.org/dialog/?doi=10.1104/pp.19.00460&domain=pdf&date_stamp=2019-11-13mailto:[email protected]://www.plantphysiol.orgmailto:[email protected]://www.plantphysiol.org/cgi/doi/10.1104/pp.19.00460

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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