Dual pathways for regulation of root branching by nitrate

Abstract

Root development is extremely sensitive to variations in nutrient supply, but the mechanisms are poorly understood. We have investigated the processes by which nitrate (NO3-), depending on its availability and distribution, can have both positive and negative effects on the development and growth of lateral roots. When Arabidopsis roots were exposed to a locally concentrated supply of NO3- there was no increase in lateral root numbers within the NO3--rich zone, but there was a localized 2-fold increase in the mean rate of lateral root elongation, which was attributable to a corresponding increase in the rate of cell production in the lateral root meristem. Localized applications of other N sources did not stimulate lateral root elongation, consistent with previous evidence that the NO3- ion is acting as a signal rather than a nutrient. The axr4 auxin-resistant mutant was insensitive to the stimulatory effect of NO3-, suggesting an overlap between the NO3- and auxin response pathways. High rates of NO3- supply to the roots had a systemic inhibitory effect on lateral root development that acted specifically at the stage when the laterals had just emerged from the primary root, apparently delaying final activation of the lateral root meristem. A nitrate reductase-deficient mutant showed increased sensitivity to this systemic inhibitory effect, suggesting that tissue NO3- levels may play a role in generating the inhibitory signal. We present a model in which root branching is modulated by opposing signals from the plant's internal N status and the external supply of NO3-.

Figure 1

Effects of localized supplies of different N sources on LR growth. (A) Effect of different KNO₃ concentrations in the “NO₃⁻-rich” zone. Arabidopsis seedlings were grown on vertical agar plates that had been divided horizontally into three segments to allow different nutrient treatments to be applied to the basal, middle, and apical zones of the primary root (12). The top and bottom segments of the agar plate contained 0.01 mM NH₄NO₃ as sole N source, and the middle segment contained in addition the indicated concentrations of KNO₃. The controls received 1 mM KCl in the middle segment (in preliminary experiments, the 1 mM KCl treatment was found to have no effect on LR growth compared with a water control). Nine days after transfer, LR lengths in the top (shaded bars) and middle (filled bars) segments were measured for each seedling (n = 23–33). (B) Effect of a localized supply of KNO₃ on LR elongation rates in the NO₃⁻-rich zone. Seedlings (12 per treatment) were grown at 25°C and under continuous light on segmented agar plates containing 0.01 mM NH₄NO₃ that were supplemented in the middle segment with either 1 mM KCl or 1 mM KNO₃. The elongation rates of individual LRs in the middle segment were estimated by measuring their lengths on days 8 and 9 after transfer, and the frequency distribution of different elongation rates was plotted for the KCl (hatched bars) and KNO₃ (filled bars) treatments. The mean rate of LR elongation in the KCl controls was 2.7 ± 0.26 and in the KNO₃ treatment was 5.4 ± 0.38 mm⋅day⁻¹. (C) Relationship between the elongation rate of a LR and the length of its mature cells. The elongation rates of individual LRs growing in a localized supply of 1 mM KCl or 1 mM KNO₃ as described for B were determined for the 24-h period before they were excised and fixed for cytological examination. Mean mature cell lengths for the KCl (○) and KNO₃ (●) treatments were estimated as described in Materials and Methods. (D) Effect of localized supplies of NH₄⁺ and glutamine on LR elongation rates. Seedlings (11–13 per treatment) were grown on segmented agar plates containing 0.01 mM NH₄NO₃ and supplied in the middle segment with KCl, KNO₃, NH₄Cl, or glutamine (each at 0.1 mM). LR elongation rates in the top (shaded bars) and middle (filled bars) segments were measured between days 9 and 10 after transfer.

Figure 2

The responses of three auxin-resistant mutants to a localized supply of KNO₃. The auxin-resistant mutants (aux1–7, axr2–1, and aux4–2) and the wild type (Col) were grown under standard low-N conditions (see Fig. 1) on segmented agar plates, and the middle segment was supplied with either 1 mM KCl (control) or 1 mM KNO₃ (localized NO₃⁻ supply). Nine days after transfer, LR lengths were measured in the top (shaded bars) and middle (filled bars) segments (13 seedlings of each line per treatment).

Figure 3

Effect of a high rate of NO₃⁻ supply on LR development. (A) Photograph showing the suppression of LR development in seedlings grown for 7 days on 50 mM KNO₃ compared with those grown on 1 mM KNO₃. (B) Close-up of a typical stunted LR from a seedling grown on 50 mM KNO₃. The primary root is approximately 0.2 mm in diameter. (C) LR development is specifically inhibited at a stage just after LR emergence. Seedlings of the END199 GUS marker line (30) were grown on 1 mM KNO₃ (shaded bars) or 50 mM KNO₃ (filled bars) for 7 days and then stained for GUS activity (9–15 seedlings per treatment). Each LR or LR primordium was classified according to its stage of development and its distance from the primary root tip. The relative frequency of each of the four developmental stages within each 1-cm segment of the primary root has been plotted. Stage A, up to 3 cell layers; Stage B, unemerged, >3 cell layers; Stage C, LR emerged, <0.5 mm long; Stage D, ≥0.5 mm long.

Figure 4

Characterization of the inhibitory effect of NO₃⁻ in wild-type and an NR-deficient line. (A) The inhibitory effect of a high NO₃⁻ concentration is systemic. Seedlings were grown on segmented agar plates in which the top and bottom segments contained either 0.01 mM NH₄NO₃ (control) or 50 mM KNO₃, whereas the middle segments received either 1 mM KCl (hatched bars) or 1 mM KNO₃ (filled bars). LR lengths in the middle segment were measured 8 days after transfer (11–17 seedlings per treatment). (B) Increasing the sucrose concentration in the medium partially relieves the inhibitory effect of high NO₃⁻ concentrations. Seedlings were grown for 7 days on unsegmented agar plates containing a range of KNO₃ concentrations and either 0.5% (open bars) or 2% (shaded bars) sucrose. Note that there was no significant LR growth in the 50 mM NO₃⁻ treatment at 0.5% sucrose. (C) An NR-deficient mutant is more sensitive than the wild type to the inhibitory effect of high NO₃⁻ concentrations. Seedlings of the wild type (Col) and of the nia1nia2 mutant (22) (12–18 per treatment) were grown for 7 days on agar plates containing a range of KNO₃ concentrations, and the numbers of LRs at stages C (shaded bars) and D (filled bars) (see Fig. 3) were scored by bathing the roots in water and examining them at ×100 magnification with an inverted microscope.

Figure 5

Dual-pathway model for regulation of LR growth and development by NO₃⁻. Because the ANR1 gene is rapidly induced by NO₃⁻ (12), the putative NO₃⁻ receptor and the mechanism for transcriptional activation of ANR1 are likely to be shared with other NO₃⁻-inducible genes such as the NIA1 genes encoding NR (39, 40). We have tentatively placed ANR1 upstream of AXR4 in the signal transduction pathway. This arrangement makes a number of predictions that can be tested experimentally by using axr4 mutants (19) and ANR1 antisense lines (12). Other genes implicated in controlling particular stages in LR initiation or development (29) are shown on the right. Broken arrows indicate signaling steps, solid arrows indicate transport or metabolic steps, and large open arrows indicate developmental steps.