PLENTY, a hydroxyproline O-arabinosyltransferase, negatively regulates root nodule symbiosis in Lotus japonicus

Abstract

Legumes can survive in nitrogen-deficient environments by forming root-nodule symbioses with rhizobial bacteria; however, forming nodules consumes energy, and nodule numbers must thus be strictly controlled. Previous studies identified major negative regulators of nodulation in Lotus japonicus, including the small peptides CLAVATA3/ESR (CLE)-RELATED-ROOT SIGNAL1 (CLE-RS1), CLE-RS2, and CLE-RS3, and their putative major receptor HYPERNODULATION AND ABERRANT ROOT FORMATION1 (HAR1). CLE-RS2 is known to be expressed in rhizobia-inoculated roots, and is predicted to be post-translationally arabinosylated, a modification essential for its activity. Moreover, all three CLE-RSs suppress nodulation in a HAR1-dependent manner. Here, we identified PLENTY as a gene responsible for the previously isolated hypernodulation mutant plenty. PLENTY encoded a hydroxyproline O-arabinosyltransferase orthologous to ROOT DETERMINED NODULATION1 in Medicago truncatula. PLENTY was localized to the Golgi, and an in vitro analysis of the recombinant protein demonstrated its arabinosylation activity, indicating that CLE-RS1/2/3 may be substrates for PLENTY. The constitutive expression experiments showed that CLE-RS3 was the major candidate substrate for PLENTY, suggesting the substrate preference of PLENTY for individual CLE-RS peptides. Furthermore, a genetic analysis of the plenty har1 double mutant indicated the existence of another PLENTY-dependent and HAR1-independent pathway negatively regulating nodulation.

Fig. 1.

Complementation of plenty. (A) Rhizobium-inoculated MG-20 plants stably transformed with the empty vector pUB-GW-GFP (ev/MG-20), a plenty mutant transformed with the empty vector (ev/plenty), and a plenty mutant transformed with pUB-GW-Full-PLENTY (PLENTY/plenty) at 14 days after inoculation (DAI) with M. loti MAFF303099. Magnified images of the nodulated regions of the ev/MG-20 (B), ev/plenty (C), and PLENTY/plenty (D) plants are shown. (E) Non-inoculated plants of ev/MG-20, ev/plenty, and PLENTY/plenty at 21 days after germination (DAG). (F) Boxplots of the nodule numbers [≥0.5 mm diameter (left), <0.5 mm diameter (middle), and total (right)] of the individual inoculated T₃ transgenic lines (n≥10). (G) Boxplots of the primary root length of the inoculated T₃ transgenic plants at 14 DAI (left) and the non-inoculated T₃ transgenic plant at 21 DAG (right). Scale bars=2 cm in (A, E) and 2 mm in (B–D). Different lower case letters represent statistically significant differences (P<0.05; Tukey’s HSD). Experiments were performed in triplicate (n≥10 in each trial).

Fig. 2.

Subcellular localization of PLENTY–GFP fusion proteins. (A) Overview of the three GFP fusion protein constructs; GFP, Full-PLENTY–GFP, and N–GFP containing the first 58 amino acids of the N-terminal region of PLENTY. PLENTY has a putative secretory signal peptide at the N-terminus (shown in blue). (B–L) Confocal microscopic images of the localization of a series of PLENTY–GFP fusion proteins driven by the CaMV 35S promoter. (B, C) The transient expression in onion epidermal cells transformed using particle bombardment. (D–L) Transient expression in N. benthamiana pavement cells co-expressing the mCherry-fused cis-Golgi marker, transformed using A. tumefaciens infiltration. The constructs used for each analysis are shown in each panel. Merged images show the cytoplasmic localization of GFP (F) and the Golgi localization of Full-PLENTY–GFP and N–GFP (I, L). Scale bars=50 μm in (B, C) and 25 μm in (D–L). Similar GFP localization was observed in >10 transformed cells.

Fig. 3.

Identification of the HPAT activity of PLENTY in vitro. (A) Western blots of microsomal proteins in yeast expressing C-terminally FLAG-fused PLENTY proteins [Full-PLENTY (41.6 kDa), ΔN2-PLENTY with the first 25 amino acids deleted (38.8 kDa), or ΔN1-PLENTY with the first 46 amino acids deleted (36.4 kDa)], probed using an anti-FLAG antibody. (B) Identification of HPAT activity of the three recombinant PLENTY proteins. The synthetic substrate peptide (PGVOOS)₃ was incubated with the FLAG-tag-fused recombinant proteins in the presence of UDP-β-l-Araf and analyzed using LC/MS. The 1849.6 increase in m/z corresponds to the arabinosylation.

Fig. 4.

Hypernodulation of plenty was strongly suppressed by CLE-RS1 and mildly suppressed by CLE-RS2 but not by CLE-RS3. (A) Stereoscopic images of transgenic hairy roots constitutively expressing GUS and LjCLE3 (as a control), LjCLE-RS1, LjCLE-RS2, or LjCLE-RS3. The constructs used for each analysis are shown in each panel. Scale bars=5 mm. (B) Boxplots of the number of nodules per individual transformed plant at 14 DAI with M. loti MAFF303099. The genotypes and introduced constructs are indicated on the graph. Statistical analyses were conducted using a two-tailed Welch’s t-test (**P<0.01, *P<0.05, n≥10). The black dots represent outliers. Experiments were performed in triplicate (n≥10 in each trial).

Fig. 5.

Additive nodulation of the plenty har1-7 double mutant. (A) Nodulation in the wild-type (MG-20), plenty, har1-7, and plenty har1-7 double mutant plants. (B–E) Magnified images of nodulated roots of the wild type (MG-20) (B), plenty (C), har1-7 (D), and the plenty har1-7 double mutant (E). (F) Boxplot of the nodule number [≥0.5 mm diameter (left), <0.5 mm diameter (middle), total (right)], normalized by the total root length of each plant, counted at 21 DAI with M. loti MAFF303099. (G) Boxplot of the lengths of lateral loots (left), primary roots (middle), and total roots (right) of each plant, measured for normalization in (F). Scale bars=2 cm in (A) and 5 mm in (B–E). The values of the total nodule numbers were used for the statistical analysis in (F). Different lower case letters represent statistically significant differences (P<0.05; Tukey’s HSD; n=14). 0.01<P<0.05 are denoted on the graph. The black dots represent outliers. Experiments were performed in triplicate (n≥10 in each trial).