TaGSK3 phosphorylates TaNLP7 to repress nitrogen deficiency‐induced leaf senescence in wheat

Bread wheat (Triticum aestivum L.) is a staple crop world-wide, contributing to c. 20% of human calories and protein consumption (Braun et al., 2010; Giraldo et al., 2019; Xiao et al., 2022). The intensive application of nitrogen fertilizer boosts crop yield but causes serious detrimental effects on ecosystems (Liu et al., 2022). It is therefore crucial to breed low-nitrogen-tolerant crop varieties to achieve a stable yield under low-nitrogen conditions (Li et al., 2018; Wu et al., 2020; Liu et al., 2021; Song et al., 2023). Nitrate is the primary nitrogen source and also a signaling molecule for the plants grown in aerobic soil. The Arabidopsis nitrate-coupled Ca2+-sensor protein kinases (CPKs) can phosphorylate the NIN-LIKE PROTEIN 7 (NLP7) transcription factor, acting as a master regulator that orchestrates the primary nitrate responses to promote its persistent nuclear localization (Liu et al., 2017). A recent study demonstrated that the NLP7 transcription factor is a plant nitrate sensor (Liu et al., 2022). Nitrogen deficiency usually causes leaf senescence of plants (Cheng et al., 2023). We previously cloned a gain-of-function allele of the GSK3/SHAGGY-like kinase-encoding gene TaGSK3 in wheat, which causes the dark-green leaf and compact plant architecture phenotypes (Dong et al., 2023). Here, we sought to investigate the effect of TaGSK3 on nitrogen deficiency-induced leaf senescence in wheat. To determine the potential effect of TaGSK3 on nitrogen starvation responses in wheat, the modern cultivar YZ4110 and the gain-of-function mutant Tagsk3 (Tagsk3E286K) in the YZ4110 background were grown under normal nitrogen (2 mM KNO3, Normal N) and low-nitrogen (0.2 mM KNO3, Low N) conditions, respectively (Fig. 1a). The results showed that the gain-of-function mutant Tagsk3E286K exhibited an attenuated low-nitrogen-induced leaf senescence phenotype compared with YZ4110 (Fig. 1a). Consistently, the Chl content of YZ4110 was markedly reduced by c. 59% after low-nitrogen treatment, whereas this reduction in the Tagsk3E286K mutant was c. 42% (Fig. 1b). Similarly, the overexpression transgenic plants of TaGSK3E285K (a gain-of-function mutated protein form of TaGSK3) in the modern cultivar KN199 background also exhibited an attenuated low-nitrogen-induced leaf senescence phenotype compared with KN199 (Fig. 1a). The Chl content of KN199 was markedly reduced by c. 63% under low-nitrogen conditions, whereas this reduction in the TaGSK3E285K OE transgenic plants was only c. 38% (Fig. 1b). Moreover, the photochemical efficiency of PSII (Fv/Fm) of Tagsk3E286K mutant and TaGSK3E285K OE transgenic plants was higher than that of their corresponding controls under low-nitrogen conditions, respectively (Fig. S1). Furthermore, we analyzed the expression patterns of wheat STAY-GREEN (TaSGR) genes in these lines, which are widely regarded as reliable markers of leaf senescence (Jiang et al., 2007; Park et al., 2007; Ren et al., 2007; Zhang et al., 2022). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays confirmed that the expression levels of TaSGR were significantly upregulated in the YZ4110 and KN199 under nitrogen deficiency conditions, whereas this induction in the Tagsk3E286K mutant and TaGSK3E285K OE transgenic plants was largely abolished (Fig. 1c). Simultaneously, we found that the nitrogen contents in the Tagsk3E286K mutant and TaGSK3E285K OE transgenic plants were also increased compared with the control YZ4110 and KN199 both under normal nitrogen and low-nitrogen conditions (Fig. 1d,e). Taken together, we conclude that TaGSK3 is involved in the regulation of nitrogen deficiency-induced leaf senescence in wheat. To explore the underlying mechanism of TaGSK3 in regulating nitrogen deficiency-induced leaf senescence, we detected the possible interaction between TaGSK3 and TaNLP7. Firefly luciferase complementation imaging assays in Nicotiana benthamiana leaves revealed that TaGSK3 could interact with TaNLP7 in plant cells (Fig. 1f). Bimolecular fluorescence complementation assays also demonstrated that TaGSK3 could interact with TaNLP7 (Fig. S2). Considering that TaGSK3 is a serine/threonine protein kinase, we wondered whether TaGSK3 phosphorylates the TaNLP7 protein. To address this question, we used the Phos-tag approach by incubating the TaNLP7N(1–400)-GST and TaNLP7C(401–934)-GST proteins together with TaGSK3-MBP in the kinase reaction buffer. The results showed that the TaNLP7N but not TaNLP7C could be phosphorylated by TaGSK3 (Fig. S3). To identify the phosphorylation sites in TaNLP7N, liquid chromatography–tandem mass spectrometry (LC-MS) assays were performed for TaNLP7N incubated with TaGSK3 in vitro. As a result, the residues Ser91, Ser94, Thr96, Thr213, Ser284 and Thr285 were identified as putative phosphorylation sites of TaNLP7 by TaGSK3 (Fig. S4). We then mutated the six putative phosphorylation sites to Ala (A) residues to generate the TaNLP7N (6A) mutated protein. The in vitro phosphorylation assays showed that the phosphorylation of TaNLP7N (6A) proteins by TaGSK3 was largely reduced (Fig. 1g), suggesting that the Ser91, Ser94, Thr96, Thr213, Ser284 and Thr285 residues are the major phosphorylation sites of TaNLP7 by TaGSK3. Next, we evaluated the effect of TaGSK3-mediated phosphorylation on TaNLP7. To this end, the TaNLP7-GFP and TaNLP7(6A)-GFP fusion proteins together with or without GSK3-Flag were transiently expressed in N. benthamiana leaves, respectively. Confocal microscopy assays showed that the TaNLP7-GFP protein was accumulated in the nucleus, whereas the TaNLP7(6A)-GFP protein was persistently accumulated in the intracellular periphery (Fig. 1h). Notably, we found that GSK3 promoted the nuclear accumulation of TaNLP7, whereas it had no effect on TaNLP7(6A) (Fig. 1h). Furthermore, we generated the TaNLP7-OE and TaNLP76A-OE transgenic wheat plants. The results showed that the TaNLP7-OE transgenic plants exhibited an attenuated nitrogen deficiency-induced leaf senescence phenotype to some extent, whereas the TaNLP76A-OE transgenic plants were similar to that of wild-type Fielder (Fig. S5a,b). Moreover, the Chl content of TaNLP7-OE transgenic plants was higher than that of the Fielder and TaNLP76A-OE transgenic plants under low-nitrogen conditions (Fig. S5c). RT-qPCR assays showed that the nitrogen deficiency-induced expression levels of TaSGR in the TaNLP7-OE transgenic plants were lower than those of the Fielder and TaNLP76A-OE transgenic plants (Fig. S5d). Notably, four putative TaNLP7 binding motifs (nitrate responsive cis-element-like, NRE-like) were contained in the TaSGR-5A promoter (Fig. S6). Chromatin immunoprecipitation (ChIP)-qPCR assays demonstrated that TaNLP7 associates with the promoter of TaSGR-5A (Fig. 2a). Transient expression assays in N. benthamiana leaves showed that TaNLP7 repressed TaSGR-5Apro:LUC expression but failed to repress TaSGR-5AmNREpro:LUC expression (Figs 2b, S6, S7). To determine the role of TaSGR in nitrogen deficiency-induced leaf senescence, we generated the Tasgr-aabbdd mutants by CRISPR/Cas9-mediated gene editing (Fig. 2c). Excitingly, the low-nitrogen-induced leaf senescence was markedly attenuated in the Tasgr-aabbdd mutants compared with Fielder (Figs 2d,e, S8). Notably, under normal nitrogen conditions, the Chl content in the TaGSK3E285K OE transgenic plant, but not in the Tasgr-aabbdd mutant, was higher than that of their corresponding controls (Figs 1b, 2e). This difference may be due to the roles of TaGSK3 in regulating both TaSGR-mediated Chl degradation and TaBZR1-regulated Chl biosynthesis (Luo et al., 2010; Oh et al., 2012; Wang et al., 2020). In summary, this study demonstrates that TaGSK3 negatively regulates nitrogen deficiency-induced leaf senescence in wheat, at least partly by phosphorylating TaNLP7, providing new insights into the molecular mechanisms underlying plant nitrogen starvation responses. Currently, developing low-nitrogen-tolerant crop varieties requiring reduced nitrogen input is an urgent goal for global sustainable agriculture. In the future, it will be important to evaluate the potential value of the gain-of-function Tagsk3 mutant, which represents a low-nitrogen-tolerant wheat line, under nitrogen-limited field conditions. We thank Prof. Xiuying Kong for providing the Tagsk3 (Tagsk3E286K) mutant in the YZ4110 background. This research was supported by the National Key Research and Development Program of China (2023YFF1000600, 2023YFF1000402, 2023YFD1200403) and Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-CSAL-202401, CAAS-BRC-CS-2025-01). None declared. JS designed the research. ZY, WB, GG and SH performed the experiments with the assistance of YW. Y. Zhou and YZ analyzed the data and revised the manuscript. ZY and JS wrote and revised the manuscript. ZY, WB, GG and SH contributed equally to this work. The data that support the findings of this study are available in the article and its Supporting Information files (Figs S1–S8; Methods S1; Table S1). Sequence data can be found in Ensembl Plants (http://plants.ensembl.org/index.html) according to accession nos.: TaGSK3 (TraesCS3D02G137200); TaNLP7 (TraesCS3A02G159600/TraesCS3B02G190300/TraesCS3D02G166900); TaSGR (TraesCS5A02G319900/TraesCS5B02G320200/TraesCS5D02G325900). Fig. S1 Photochemical efficiency of PSII (Fv/Fm) in the leaves of Tagsk3E286K mutant and TaGSK3E285K OE transgenic plants under normal and low-nitrogen conditions. Fig. S2 Bimolecular fluorescence complementation assays in Nicotiana benthamiana showing the interaction of TaGSK3 and TaNLP7. Bars, 25 μm. Fig. S3 Protein phosphorylation assays for the TaNLP7N and TaNLP7C truncated proteins by TaGSK3 using Phos-tag gel. Fig. S4 Phosphorylation sites of TaNLP7N by TaGSK3 determined by LC-MS/MS. Fig. S5 Functional characterization of TaNLP7 in low N-induced wheat leaf senescence. Fig. S6 Schematic diagram of the TaSGR-5Apro and TaSGR-5AmNREpro promoters with nitrate responsive cis-elements-like (NRE-like) or mutated NRE-like motifs. Fig. S7 TaNLP7 could not repress the expression of the reporter TaSGR-5AmNREpro:LUC in the Nicotiana benthamiana leaves. Fig. S8 The photochemical efficiency of PSII (Fv/Fm) in the leaves of Tasgr-aabbdd mutants under normal and low-nitrogen conditions. Methods S1 Materials and methods. Table S1 Primers used in this study. Please note: Wiley is not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. 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