CYTOKININ OXIDASE/DEHYDROGENASE3 Maintains Cytokinin Homeostasis during Root and Nodule Development in Lotus japonicus

Abstract

Cytokinins are required for symbiotic nodule development in legumes, and cytokinin signaling responses occur locally in nodule primordia and in developing nodules. Here, we show that the Lotus japonicus Ckx3 cytokinin oxidase/dehydrogenase gene is induced by Nod factor during the early phase of nodule initiation. At the cellular level, pCkx3::YFP reporter-gene studies revealed that the Ckx3 promoter is active during the first cortical cell divisions of the nodule primordium and in growing nodules. Cytokinin measurements in ckx3 mutants confirmed that CKX3 activity negatively regulates root cytokinin levels. Particularly, tZ and DHZ type cytokinins in both inoculated and uninoculated roots were elevated in ckx3 mutants, suggesting that these are targets for degradation by the CKX3 cytokinin oxidase/dehydrogenase. The effect of CKX3 on the positive and negative roles of cytokinin in nodule development, infection and regulation was further clarified using ckx3 insertion mutants. Phenotypic analysis indicated that ckx3 mutants have reduced nodulation, infection thread formation and root growth. We also identify a role for cytokinin in regulating nodulation and nitrogen fixation in response to nitrate as ckx3 phenotypes are exaggerated at increased nitrate levels. Together, these findings show that cytokinin accumulation is tightly regulated during nodulation in order to balance the requirement for cell divisions with negative regulatory effects of cytokinin on infection events and root development.

Figure 1.

Lotus japonicus CKX family. A, CKX phylogeny assembled by alignment of the L. japonicus, M. truncatula, and Arabidopsis amino acid sequences. LjCKX3 (red) is most closely related to two M. truncatula genes, which are induced by Nod factor application (van Zeijl et al., 2015). Bootstrap values are shown for each node based on 1,000 replications. B, LjCkx3 comprises five exons and encodes a predicted signal peptide (SP), cytokinin (CK bind), and FAD binding domains. Lines containing LORE1 insertions were characterized in the first and third exons of LjCkx3.

Figure 2.

Effect of ectopic Nod factor application or M. loti inoculation on Ckx3 mRNA levels. A, Relative expression levels following Nod factor treatment. B, Relative expression levels following inoculation with M. loti R7A. Values are relative to mock treatment and indicate mean ± 95% CI for n = 3. P-values were calculated using Wilcoxon rank-sum testing between mock and treatment groups and are indicated by *<0.05.

Figure 3.

Activity of LjCkx3 1 kb promoter during root and nodule development. Spatio-temporal expression driven by the Ckx3 promoter was determined in hairy roots using confocal microscopy with a LjCkx3::tYFPnls reporter (nuclear-localized, green). A to C are uninoculated, while d to H are inoculated with M. loti expressing DsRED (magenta) A, The meristematic zone at the root tip independent of inoculation. B, Expression in uninoculated roots. C, Root cross-section showing expression in pericycle adjacent to xylem poles and in protoxylem. D, Expression in the central vascular cylinder of inoculated roots. E, Nodule primordia at 5 d after inoculation with M. loti. Note the expression associated with dividing cells in the cortex and pericycle. F, Nodule primordia 5 dpi. G, Cortex of growing nodules. H, Periphery of fixing nodules. A, B, and D are whole mounts, while others show sections (80–100 µM). C, Cortex; E, endodermis; P, pericycle; X, xylem; Ep, epidermis; IT, Infection Thread; V, Vascular cylinder. Bar, 100 µM.

Figure 4.

Cytokinin free base and riboside levels in L. japonicus roots following inoculation with M. loti. A, trans-Zeatin. B, trans-Zeatin Riboside. C, cis-Zeatin. D, cis-Zeatin Riboside. E, Dihydrozeatin. F, Dihydrozeatin Riboside. G, Isopentenyladenine. H, Isopentenyladenine Riboside. Concentrations were measured in mock (white bars) M. loti R7AnodC (striped bars) and M. loti R7A (checked bars) treated Lotus japonicus Gifu and ckx3-2 whole roots 24 h and 72 h after treatment as indicated. Stars represent comparisons to R7AnodC for wild-type bars and between ckx3-2 and wild type under the same treatment group for ckx3-2 bars. Values represent mean ± 95% CI for n = 4 biological replicates. P-values as determined by Wilcoxon rank-sum testing are indicated by *<0.05.

Figure 5.

Symbiotic phenotypes of Ljckx3 mutants. A, Number of nodules per plant 7 to 14 d after inoculation for Gifu, ckx3-1, and ckx3-2 grown on agar slants supplemented with 1 mm KNO₃ and inoculated with M. loti R7A. B, Number of infection threads formed per root 10 d after inoculation with M. loti expressing DsRED. C, Number of infection threads formed on plants grown on agar slants supplemented with AVG (10⁻⁸ M) 10 d after inoculation with M. loti expressing DsRED. D, Number of infection threads formed on plants grown in the presence of nitrate and BAP (10⁻⁸ M). Values represent mean ± 95% CI for n = 30–49 for nodule counts and n = 9 to 10 for IT counts. Statistical comparisons are shown between wild type and ckx3 mutants in A and B and between treatments and control for each genotype in C and D. P-values were calculated using ANOVA and Tukey posthoc testing and are indicated by *<0.05, **<0.01, ***<0.001.

Figure 6.

Sensitivity to nitrate regulation of nodulation in ckx3 mutants. A, Number of red Fix⁺ nodules (red bars) and white Fix^- nodules (white bars) formed on Gifu (plain bars) and ckx3-2 (patterned bars) roots 11 d after inoculation with M. loti R7A. Plants were grown on agar slants supplemented with the indicated KNO₃ or BAP concentrations. B and C, Box plots of acetylene reduction assay measurements conducted on whole roots (B) or individual nodules (C) 14 dpi in same conditions as above. D to I, Indicative photographs of Gifu and ckx3-2 plants under different conditions. D and E, Nitrate-free conditions. F and G, 5 mm KNO₃. H and I, 10⁻⁸ M BAP. J to O, nodule sections of Gifu and ckx3-1 grown under different conditions and inoculated with M. Loti MAFF303099 DsRED. J, Gifu nitrate free. K, Gifu 5 mm KNO_3. L, Gifu 10⁻⁸ M BAP. M. ckx3-1 nitrate free. N, ckx3-1 5 mm KNO₃. O, ckx3-1 10⁻⁸ M BAP. Values represent mean ± 95% CI for n = 16–29 for nodule counts. ARA was conducted on n = 6–9 roots or nodules. P-values in A indicate comparison of red nodule numbers and were calculated using ANOVA and Tukey posthoc testing. P-values in B and C indicate comparison of Gifu and ckx3-1 for each treatment and were calculated using Wilcoxon rank-sum testing and are indicated by *<0.05.

Figure 7.

Root development phenotypes of ckx3 mutants. A, Root length 20 d after germination. B, Length from the root tip to the first emerging root hair. C, Root hair development determined by the angle enclosing emerging root hairs (see Supplemental Fig. S4). Values represent mean ± 95% CI for n = 27–34 for root lengths and n = 15 for root tip and root hair measures. P-values were calculated using ANOVA and Tukey posthoc testing and are indicated by *<0.05, **<0.01, ***<0.001.