Fig. 4: The A. thaliana (Arabidopsis) miR2111-HOLT regulon controls lateral root (LR) initiation at moderate nitrate starvation.

a, b Stably transformed A. thaliana (Arabidopsis) plants expressing pMIR2111b:GUS show predominant GUS activity in the phloem of leaf veins. b Leaf cross section of pMIR2111b:GUS plants. ph, phloem and xy, xylem. c Numbers of LR initiations at different nitrate concentration in Arabidopsis wild type (Col-0) plants. d, e miR2111 (d) and HOLT (e) levels in Col-0 and p35s::MIR2111b expressing plants (2111ox) at 1 mM nitrate. f LR initiations in holt-1, holt-2 and 2111ox plants compared to Col-0. g Schematic model of nitrate responsiveness of Arabidopsis lateral root initiations. holt-1 and 2111ox plants lack a foraging response at moderate nitrate starvation. h, i miR2111 (h) and HOLT (i) levels of Col-0 at varying nitrate concentrations. j Simplified model outlining nitrate dependency of miR2111 and HOLT levels, and of root architectural responses in Arabidopsis between 1 and 10 mM nitrate. k Number of LR initiations on 2111ox / Col-0 (shoot / root) grafts compared to Col-0 / Col-0 control grafts at 1 mM KNO₃. l Combined simplified model of Arabidopsis and L. japonicus (Lotus) root responses to varying nitrate supply. HOLT positively correlates with LR initiations in both species, but nitrate dependent abundance patterns of both LR initiations and TML/HOLT levels are opposite. d, e, h, i qRT-PCR analyses. RNA levels are relative to those of two reference genes, whole plant tissue harvested 10 days after germination. c, f, k Analysis seven days after germination (c, f) or after graft regeneration (k). Comparisons used analysis of variance (ANOVA) and post-hoc Tukey testing (p ≤ 0.05), with distinct letters indicating significant differences (c, f, h, i) or Student’s t-test (*p ≤ 0.05) (d, e, k). a, b Scale bars equal 200 µm (a) or 100 µm (b). a, b all 21 tested plants of 3 independent lines showed a similar expression pattern. Analysis of three independent lines showed similar results. c, f, h, i Plants were grown at indicated nitrate concentrations using ½ strength MS media free of other nitrogen sources. c, f–j, l Trendlines are simplified and not to scale. Sample size, replicates and exact p-values are listed in the Source Data file. Dotplots show individual data points and a line indicating their average value. Boxplot central line shows median value, box limits indicate the 25th and 75th percentile. Whiskers extend 1.5 times the interquartile range, or to the last datapoint. Individual datapoints are represented by dots.