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Literature Sharing | The TaCLE24b peptide signaling cascademodulates lateral root development and drought tolerance in wheat
Release time:
2025-03-05
Plant peptide hormones play an important role in regulating root development and stress responses. Here, we identify a root-derived CLAVATA3/Endosperm surrounding region-related (CLE) peptide TaCLE24b and its leucine-rich repeatreceptor-like kinase CLAVATA1 (CLV1) TaCLV1, which together regulate theformation of lateral root primordium and the outgrowth of lateral root inwheat. Additionally, a loss-of-function mutation of TaSG-D1, encoding a Ser/Thr protein kinase glycogen synthase kinase 3 (STKc_GSK3), also enhances theformation of lateral root primordium and the outgrowth of lateral roots in wheat. Furthermore, TaCLV1 interacts with TaSG-D1, leading to phosphorylation and degradation of TaSG-D1. Whereas the presence of TaCLE24b enhances the binding affinity and phosphorylation ability of TaCLV1 to TaSG-D1,thereby positively fine-tuning lateral root development and contributing to drought tolerance in wheat. Overall, our findings provide new insights into the significance of the peptide signaling pathway in regulating lateral root development and responding to drought stress in wheat.
Previous study has predicted 104 CLE genes in wheat genome, andnearly half of which exhibited higher transcript abundance in wheat roots than in other tissues according to the published transcriptomedata15, indicating their potential roles in the regulation of root development. Of these, TraesCS5B02G418500 (hereafter designed asTaCLE24b according to the previous study15) was mainly expressed in seedling root according to RT-qPCR analysis, whereas its homeologs of TaCLE24a and TaCLE24d are barely expressed in all examined tissues(Fig. 1a and Supplementary Fig. 1a). Therefore, we selected TaCLE24b as the candidate for further analysis. Further β-Glucuronidase (GUS) reporter-aided histochemical analysis demonstrated that the TaCLE24b promoter is predominantly active in the lateral root primordia and root tips of wheat (Fig. 1b), which was further confirmed by in situ hybridization (Supplementary Fig. 1b).
Fig. 1 | TaCLE24b positively contributes to lateral root development in wheat
Peptide hormones are often recognized by corresponding receptors,which then transmit signals to downstream targets to fine-tune plant growth or stress responses17–19. To identify potential candidate receptors involved in the TaCLE24b peptide signaling pathway in wheat, we screened a subset of leucine-rich repeat receptor-like kinases (Methods, Supplementary Fig. 2b), and selected TraesCS7D02G521200(designed as TaCLV1), a homolog of Arabidopsis CLV1, for subsequent analysis. TaCLV1 exhibited a similar expression pattern to TaCLE24b,and was mainly expressed in lateral root primordium and root tip based on RT-qPCR and in situ hybridization (Fig. 2a, b and Supplementary Fig. 2c). The TaCLV1 protein localized to the plasma membrane, as shown by transient expression analysis in Nicotiana benthamiana leaves (Supplementary Fig. 2d). We then demonstrated that mature TaCLE24b peptide interacted with leucine-rich repeat(LRR) domain of TaCLV1 via microscale thermophoresis (MST) assay and obtained the calculation of the dissociation constant (Kd value) of 19.35 ± 5.03 nM (Fig. 2c).
Fig. 2 | Receptor-like kinase TaCLV1 regulates lateral root development via interacting with the TaCLE24b peptide.
We previously conducted map-based cloning of TaSG-D1(TraesCS3D02G137200) gene, a homolog of Arabidopsis BRASSINOSTEROID-INSENSITIVE2 (BIN2). It was discovered that a single amino acid substitution (E286K) of TaSG-D1 (referred to as TaSG-D1E286K) results in semispherical grain phenotype in Triticum sphaerococcum20, and increases protein stability under normal conditions or in response to heat stress21. Here, we compared the lateral root development of near-isogenic lines that differed at the TaSGD1E286K (designated as NIL-K) or TaSG-D1 (designated as NIL-E) locus,and observed that the NIL-K lines exhibited a significant reduction of density of ELR and DLRP compared with NIL-E (average of 0.773 [NILK] vs. 1.396 [NIL-E] for ELR, and average of 1.244 [NIL-K] vs. 1.672 [NILE] for DLRP, ND4332 background; Fig. 3a, b). In addition, we found that EMS-induced loss-of-function mutation of TaSG-D1E286K (designated as M2-1) increases the density of ELR and DLRP by 111.6% and 56.8% compared to the WT (average of 1.697 [M2-1] vs. 0.802 [ND4332] for ELR, average of 1.634 [M2-1] vs. 1.042 [ND4332] for DLRP, ND4332 background, Fig. 3a and Supplementary Fig. 3a).
Similarly, the E286K substitution in the TaSG-D1 homeolog (TraesCS3A02G136500, located on chromosome 3A, designated as A236) also led to reduced density of ELR and DLRP compared to the WT (average of 1.130 [A236] vs. 1.638 [NC4] for ELR, average of 0.846[A236] vs. 1.204 [NC4] for DLRP, NC4 background, Fig. 3a and Supplementary Fig. 3b). Additionally, we investigated the lateral root development of NIL-K and NIL-E in response to TaCLE24b peptide treatment. Although both NIL-E and NIL-K lines exhibited increased lateral root density under peptide conditions, however, the NIL-K lines showed decreased responses compared to NIL-E, as the average density of LPRs plus LRs increased from 1.428 to 2.061 for NIL-E(44.3% increase), but from 0.976 to 1.316 for NIL-K (34.8% increase; Supplementary Fig. 3c). Furthermore, we generated knockout mutants with loss-of-function of A-, B- and D-homeolog of TaSG-D1(Tasg-d1cr-6 and Tasg-d1cr-9), which showed 54.2% and 44.4% increases in ELR density, respectively, compared to the control(average of 1.416 and 1.326 vs. 0.918 for WT [Fielder]), as well as ~66.6% and ~52.2% increases of DLRP density, respectively, compared to the WT (average of 1.696 and 1.549 vs. 1.018 for WT [Fielder];Fig. 3c, d and Supplementary Fig. 3d). Moreover, the TaSG-D1 loss-offunction mutants showed almost complete insensitivity to TaCLE24b peptide treatment (Fig. 3e). Thus, the results collectively suggest that TaSG-D1 functions downstream of TaCLE24b, and is a negative regulator of primordium formation and lateral root outgrowth in wheat.
Fig. 3 | TaSG-D1 affects lateral root development via interacting with TaCLV1 in wheat.
Further analysis revealed that TaCLV1 interacts with TaSG-D1, as evidenced by pull-down, split-luciferase complementation imaging(split-LUC), co-immunoprecipitation (Co-IP) and yeast two-hybrid assay (Y2H) assays (Fig. 3f–i). This interaction leads to phosphorylation and consequently degradation of TaSG-D1 according to phos-tag mobility shift assay and protein degradation analysis, whereas the proteasome inhibitor MG132 impeded the TaCLV1-mediated degradation of TaSG-D1 (Fig. 4a–c). Furthermore, split-LUC assays showed that TaCLV1 exhibited a stronger interaction with TaSG-D1 than TaSGD1E286K (Supplementary Fig. 4a). In addition, cell-free assays showed that a faster degradation rate of TaSG-D1 mediated by TaCLV1 in comparison to TaSG-D1E286K (Supplementary Fig. 4b).
Fig. 4 | TaCLV1 interacts with, and phosphorylates TaSG-D1.
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