Drought-dependent regulation of cell coupling in Arabidopsis leaf epidermis requires plasmodesmal protein NHL12

Abstract

The cytoplasm of most plant cells is connected by membrane-lined cell wall channels, the plasmodesmata (PD). Dynamic regulation of sugar, hormone, and protein diffusion through PD is essential for plant development and stress responses. Understanding this regulation requires knowledge of factors and mechanisms that control PD permeability through the modulation of callose levels in the cell wall around PD openings. We investigated PD regulation in leaf epidermal cells in relation to drought stress in Arabidopsis. PD-mediated cell wall permeability was decreased by drought stress and the hormone abscisic acid (ABA), and we tested how this related to several PD-associated genes with drought-responsive expression. Mutants of NON-RACE SPECIFIC DISEASE RESISTANCE/HIN1 HAIRPIN-INDUCED-LIKE 12 (NHL12) showed relatively low PD permeability that was unaffected by drought or ABA treatment. Overexpression of NHL12 in Nicotiana benthamiana epidermal cells increased PD permeability. Moreover, we showed that NHL12 can potentially interact with the callose synthase regulator NHL3 and we explored the effect of NHL12 abundance and/or lower interface permeability on ABA signaling genes. Our results indicate that NHL12 is a drought-responsive negative regulator of PD callose levels and, thereby, interface permeability. Results are discussed in relation to PD function during drought stress and the regulation of intercellular transport.

Keywords: ABA; callose; cell coupling; cell wall; drought signaling; drought stress; intercellular transport.

Fig. 1.

Interface permeability in leaf epidermal cells under control conditions, drought-stress conditions, and after abscisic acid (ABA) application in 3-week-old wild-type Arabidopsis plants. (A) Bright field images and images showing fluorescent tracer distributions before and after activation as well as drawings indicating the target cell area (black) and two neighboring cells (blue and green). During activation, tracer becomes fluorescent in the target cell (T) and diffuses to neighboring cells (N). St: Stomata. Scale bars 50 µm. (B) Average flux over target–neighbor interfaces indicated in (A). Flux was measured as the neighbor cell fluorescence concentration divided by interface length and activation time. (C) Plasmodesmata-mediated interface permeability, calculated as tracer flux divided by the tracer concentration potential across the interface. Asterisk indicates significant difference from control according to Student’s t-test (P<0.05). Boxes represent quartiles, × represents mean, and bars indicate 95% confidence intervals. Photoactivation experiments were carried out on at least three leaves. n=40 (control), 25 (drought), 31 (ABA).

Fig. 2.

Plasmodesmal structure in leaf epidermal cells under control and drought-stress conditions in 3-week-old wild-type plants. (A) Images showing aniline blue-stained callose at plasmodesmata. (B) Quantification of aniline blue fluorescence intensity at plasmodesmata. (C) Transmission electron micrographs of plasmodesmata. (D) Quantification of plasmodesmal neck diameter based on TEM images. (E) Plasmodesmal density per wall length. Asterisk indicates significant difference from control according to Student’s t-test (P<0.05). Boxes represent quartiles, × represents mean, and bars indicate 95% confidence intervals. n>120 (B), 36 (D), 5 (E). Scale bars 50 µm (A), 100 nm (C).

Fig. 3.

Interface permeability in leaf epidermal cells of 3-week-old mutant plants under control and drought-stress conditions. (A) Example images of nhl12-1 leaf epidermal cells showing fluorescent tracer distributions before and after activation. Bright field images are used for orientation. Regions of interest drawing indicate cell outlines of the target cell (T), in which fluorescence of the tracer was activated, and two neighboring cells (N). (B) Flux (measured here as fluorescence concentration in neighbor cell after activation divided by time of activation and interface length) into two neighbor cells indicated in (A). (C) Interface permeability in three nhl12 mutants. (D) Interface permeability in at4g16380 and at4g27450 mutants. Asterisks indicate significant difference from control conditions according to Student’s t-test (P<0.05). Boxes represent quartiles, × represents mean, and bars indicate 95% confidence intervals. Experiments were conducted on three plants for each line, with several photoactivation series conducted per leaf; n>11. ABA, abscisic acid.

Fig. 4.

Plasmodesmata in Nicotiana benthamiana epidermal cells expressing 35S:NHL12-mCherry. (A) Fluorescence image of cells expressing 35S:NHL12-mCherry. (B) Magnification of 35S:NHL12-mCherry (red), callose-specific dye aniline blue (blue), and an overlay of the two channels. (C) Quantification of aniline blue fluorescence at aggregates. (D) Bright field images and images showing fluorescent tracer distributions before and after activation. Scale bars 100 µm. (E) Plasmodesmata-mediated cell wall permeability. Asterisk indicates significant difference from control according to Student’s t-test (P<0.05). Boxes represent quartiles, × represents mean, and bars indicate 95% confidence intervals; n=20.

Fig. 5.

Plasmodesmal structure in leaf epidermal cells of 3-week-old nhl12 mutant plants. (A) Images of nhl12-1 showing aniline blue-stained callose at plasmodesmata. (B) Quantification of aniline blue fluorescence intensity at plasmodesmata. We measured 70 aniline blue dots in at least 10 cells in three plants. (C) Transmission electron micrographs of plasmodesmata. (D) Quantification of plasmodesmal neck diameter based on TEM images. In box plots, boxes represent quartiles, × represents mean, line indicates median, and bars indicate 95% confidence intervals. Significant differences between control and drought conditions were evaluated with Student’s t-test (P<0.05). n≥40 (B), n≥36 (D), n=5 (E). Scale bars 50 µm (A), 100 nm (E).

Fig. 6.

Analysis of NHL12 protein–protein interaction potential with Förster resonance energy transfer–fluorescence lifetime imaging microscopy (FRET-FLIM). All fluorescent protein fusion constructs were transiently expressed under control of the 35S promoter in N. benthamiana epidermal cells. (A–C) Images of control samples showing expression of single constructs via mTourqoise2 (mT2) fluorescence intensity in grayscale and mT2 lifetime color-coded. Scale bars 20 µm. (D–G) Images of samples expressing protein pairs labeled with mT2 and yellow fluorescent protein (YFP) showing fluorescence intensity (grayscale) or mT2 lifetime (color-coded). Interaction is indicated by a shortening of mT2 lifetime (here a shift from green towards blue), the result of energy transfer from mT2 to YFP when both fluorophores are in close proximity. Scale bars 10 µm. (H) Quantification of mT2 lifetimes. For cells expressing two constructs, lifetimes were measured at the sites of co-localization and averaged per image. Short lifetimes indicate protein–protein interaction. Significance of differences of two-construct-samples to the corresponding single-construct-sample was tested with Student’s t-test (P<0.05) and indicated by asterisk. n>5.

Fig. 7.

Characteristics of nhl12 mutant plants. (A) Images showing wild-type and nhl12 plants grown under control and drought-stress conditions for 11 d (22 d after germination). (B) Relative soil water content during the drought stress experiments. (C) Daily quantification of rosette diameters. Lines connect mean values. Shading indicates 95% confidence intervals. Three experiments were conducted and data combined. n=9 (B), 17 (C).

Fig. 8.

Transcript levels of genes related to the drought stress response measured under control conditions in 3-week-old nhl12 mutant plants relative to the transcript levels in wild-type plants. Boxes represent quartiles, line represents median, and bars indicate 95% confidence intervals. Asterisks indicate significant difference between nhl12-1 and wild type (P<0.05) by t-test. n=4. ABI1, ABSCISIC ACID INSENSITIVE 1; DREB2A, DRE-BINDING PROTEIN 2A; GPX6, GLUTATHIONE PEROXIDASE 6; HB7, HOMEOBOX-7; NCED, NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3.

Fig. 9.

Illustrations of potential functions of NHL3 and NHL12 in callose synthesis. (A) Complex formation of NHL3, PDLP5, and callose synthase CALS1 enhances callose formation at plasmodesmal necks (blue shading), thereby reducing permeability. (B) Binding of NHL12 to NHL3 might prevent activation of CALS1 and, thereby, callose formation and a reduction in permeability. In this way, the balance of NHL3 and NHL12 could finetune callose synthesis at PD. Protein structures are shown as predicted by AlphaFold (Jumper et al., 2021). NHL, NON-RACE SPECIFIC DISEASE RESISTANCE/HIN1 HAIRPIN-INDUCED-LIKE protein; PDLP, PLASMODESMATA-LOCATED PROTEIN; CALS1, CALLOSE SYNTHASE 1.