Plant Cell-Cell Transport via Plasmodesmata Is Regulated by Light and the Circadian Clock

Abstract

Plasmodesmata (PD) are essential for plant development, but little is known about their regulation. Several studies have linked PD 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 the day, but light is not sufficient to increase rates of PD transport at night, suggesting a circadian gating mechanism. Silencing expression 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 is dynamically regulated during the day/night cycle. Due to the many roles of PD in plant biology, this discovery has strong implications for plant development, physiology, and pathogenesis.

Figure 1.

PD transport is higher during the day than at night. A, To measure rates of PD transport, we used a quantitative GFP movement assay. A very low inoculum of A. tumefaciens cells (less than 100 bacterial cells) was gently infiltrated by syringe into N. benthamiana leaves so that a handful of individual epidermal cells were transformed to express monomeric GFP. GFP spread to neighboring epidermal cells was then quantified 48 h after infiltration (or as indicated in each experiment). Illustrated examples of GFP movement are shown here. Movement is scored by counting the number of neighboring cells to which GFP has moved (dark green) from the transformed cell (bright green). B, Representative confocal microscopy images of the GFP movement assay. GFP fluorescence is brightest in the nuclei in cells neighboring the transformed cell. Bar = 100 μm. C, N. benthamiana leaves were agroinfiltrated 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 infiltrated at dawn (bottom), GFP movement somewhat increased during the second night but dramatically increased during the third day. *, P < 10⁻³ and **, P < 10⁻⁵; n.s., not significant.

Figure 2.

A and B, PD callose deposition is not significantly different between day and night. Fluorescence intensity of Aniline Blue-stained leaves was assayed using confocal scanning laser microscopy in the leaf epidermis of N. benthamiana collected 3 h after dawn or 3 h after dusk. Callose levels were slightly lower at night than during the day (n = 8 leaves, P = 0.08). a.u., Arbitrary units; ns, not significant. Bar = 100 μm. C, Reducing ATP availability by silencing AtpC with VIGS significantly increased the rate of GFP transport (n ≥ 21 transformed cells; *, P = 0.003), demonstrating that light-dependent chloroplast ATP synthesis and PD transport do not positively correlate. D, Representative images of the GFP movement assay in mock (TRV::GUS) and atpC (TRV::AtpC) plants. Bar = 100 μm.

Figure 3.

PD transport is regulated by the circadian clock. Light treatment is represented by yellow, dark and treatment is represented by blue. Line i of both A and B shows the day/night cycles that would be experienced by plants if they were not transferred to new light regimes. A, After growing in 16-h-light/8-h-dark cycles, N. benthamiana leaves were agroinfiltrated at dawn or dusk (as indicated by red infiltration arrows) to express GFP. GFP movement was then assayed after 48 h (as indicated 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 significantly decreased PD transport (**, P < 10⁻²³; ns, not significant). B, PD transport was assayed in mock-treated plants (TRV::GUS) that had been growing in 12-h-light/12-h-dark cycles and then transferred to constant dark conditions; GFP movement was assayed 72 h after agroinfiltration. Subjective days and nights are shown in line i. Treatment with 12 h of light during the second subjective day after agroinfiltration (line iii) strongly increased PD transport compared with PD transport in constant dark (line ii), but treatment with 12 h of light during the second subjective night (line iv) had no significant effect on PD transport. C, Leaves of mock-treated plants (left) or TRV::LHY knockdowns (right) under 12-h-light/12-h-dark photoperiod conditions were agroinfiltrated to express GFP at subjective dawn. Plants were transferred 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 applied during subjective night or subjective day, significantly increasing PD transport only after exposure to light during the day. TRV::LHY knockdowns did not distinguish between subjective night or subjective day, significantly increasing PD transport after exposure to light during either time period. **, P < 10⁻⁷; ns, not significant.

Figure 4.

A, Leaf emergence was recorded every day for N. benthamiana plants grown under 12- or 16-h daylengths. The age of each leaf (in number of days) in the mature plant at the time of the GFP movement assay (6 weeks after germination) is shown. Cotyledons are not included in this diagram because they senesced within the first 6 weeks of growth. B, Average GFP movement in leaves of different ages is shown for plants grown with 12-h daylengths (purple boxes) or 16-h daylengths (orange boxes). GFP movement was assayed 48 h after agroinfiltration with a low inoculum of A. tumefaciens that transformed cells to express GFP. PD transport declines in the same linear relationship with leaf age in both sets of plants (n ≥ 59 transformed cells, ANCOVA P = 0.27, homogeneity of regressions P = 0.25). The linear relationship is depicted with a gray dashed line with slope −3.2 and y intercept 92.