Fig. 7: Modified Ca²⁺ spiking and IT cytoplasmic environment in the absence of MtAnn1.

a–c Transgenic roots expressing the NR-GECO1 Ca²⁺ sensor were generated to monitor nuclear Ca²⁺ oscillations in ann1-3 mutant or wild-type (R108S) plants. Variations in Ca²⁺ ion concentration, reflected by changes in the relative intensity of NR-GECO1 fluorescence, are expressed as signal-to-noise ratio (SNR, cf. ‘Methods’ section). Box plots represent average amplitude of spikes (spike SNR), calculated separately for each nucleus of root hairs with entrapped rhizobia (RHE) or with a growing IT (IT) in (a) R108S (RHE, n = 7; IT, n = 4) or in (b) ann1-3 (RHE, n = 15; IT, n = 9) or in S. meliloti-responsive root hairs in the close competence zone (c) (R108S, n = 160 and ann1-3, n = 156) at 1–4 dpi. Box plots show the distribution of values (open circles or black dots) obtained from three independent experiments. First and third quartiles (horizontal box edges), minimum and maximum (outer whiskers), median (centerline), mean (solid black circle) and outliers (crosses) are shown. Asterisks indicate statistical difference in RHE vs. IT in R108S RHs in a (p = 0.0285, one-tailed Student t-test) and in RHs of R108S vs. ann1-3 in (c) (p = 0.0018, two-tailed Mann-Whitney test). Differences were not significant in RHE vs. IT in ann1-3 RHs in (b) (p = 0.1735, one-tailed Student t-test). (d-g) TEM analysis of infected cells from apical zone II of R108S (d, e, e’) or ann1-3 (f, g, g’) were performed in 80 nm sections of 14–16 dpi nodules (n = 5 R108S nodules and n = 4 ann1-3 nodules derived from 2 independent experiments). ITs (arrows) in wild-type R108S are embedded in vesicles (yellow arrowheads) and ER-rich (blue arrowheads) cytoplasmic bridges, which are less visible in ann1-3. e’, g’ are complementary inverted LUT images of (e-g) that were generated by ImageJ. Scale bars: d, f = 5 µm, e, e’, g, g’ = 2 µm. See also Supplementary Fig. 15. Source data are provided as a Source Data file.