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. 2026 Oct 5;127(6):e71139. doi: 10.1111/tpj.71139

SnRK2.10 in apple positively regulates the nitrogen use efficiency via modulating the nitrogen uptake

Ying Ye 1, Zisheng Zuo 1, Zhaoting Li 1, Lin Luo 1, Xiaojun Huang 1, Yuduan Ding 1, Fengwang Ma 1,✉, Xiaoqing Gong 1,✉
PMCID: PMC13639512  PMID: 42834731

SUMMARY

Sucrose non‐fermentation‐1–related protein kinase 2 (SnRK2) family members play key roles in plant adaptation to environmental stress. In this study, low nitrogen (N) stress induced the expression of SnRK2.10, which mitigated the adverse effects of N deficiency in apple plants. SnRK2.10‐overexpressing (OE) plants showed enhanced growth and higher nitrogen use efficiency (NUE), whereas SnRK2.10‐silenced (Ri) plants exhibited more severe growth inhibition and reduced NUE, compared with the wild‐type (WT) plants under low N conditions. The transcriptomic and proteomic analyses indicated that SnRK2.10 regulates N metabolism and transport. It physically interacted with the nitrate transporter NRT2.5 and the ammonium transporter AMT1.1. Consistently, OE plants displayed higher NO3 − and NH4 + uptake rates than WT and Ri plants under low N stress. Functional analyses using transgenic apple lines with different expression levels of NRT2.5 or AMT1.1 demonstrated that both transporters positively regulate growth and NUE in apple plants' response to low N stress. Co‐expression of SnRK2.10 with either NRT2.5 or AMT1.1 effectively altered plant growth under low N stress. Collectively, these results indicate that SnRK2.10 positively regulates plant growth and NUE under low N stress, probably by promoting N uptake through direct interaction with key N transporters.

Keywords: apple; nitrogen stress, SnRK2.10, transport protein; NUE

Significance Statement

Boosting nitrogen use efficiency in perennial fruit crops sustains yields and cuts fertilizer inputs and environmental harm. This study reveals that SnRK2.10 mediates apple adaptation to low‐nitrogen conditions via regulating nitrogen uptake, providing valuable genetic resources for breeding cultivars with high nitrogen use efficiency.

INTRODUCTION

Nitrogen (N) is an essential nutrient for plant growth and development and a major component of amino acids, enzymes, and diverse metabolites (Perchlik & Tegeder, 2018). It regulates multiple aspects of plant metabolism, growth, and development, including seed germination (Cheng et al., 2021), root system establishment (Ma et al., 2014), organ development (Li et al., 2024), starch accumulation (Ullah et al., 2023), and secondary metabolism (Comadira et al., 2015). Adequate N supply enhances photosynthesis, crop yield, and root growth (Sadras & Richards, 2014), whereas N deficiency severely restricts plant growth and development (Huché‐Thélier et al., 2011). To adapt to low‐N conditions, plants have evolved coordinated morphological, physiological, and molecular responses. For example, in Arabidopsis, nitrate‐regulated root growth is mediated by ABA accumulation at the root tip to maintain meristem activity (Ondzighi‐Assoume et al., 2016). Under limited nitrate availability, ABA also improves nitrate uptake by regulating the expression of NRT2/NAR genes (Wang et al., 2020). In apple rootstocks, the transcription factor bHLH130 modulates N uptake under low‐N stress by regulating flavonoid and lignin biosynthesis (Wang et al., 2023).

Sucrose non‐fermenting 1‐related protein kinases (SnRKs) are major protein kinases involved in plant signal transduction and are key components of cascade signaling pathways. They function as central regulators of cellular energy and metabolic balance (Boudsocq et al., 2004; Robaglia et al., 2012) and serve as hubs integrating nutrient metabolism, energy status, and stress‐response signaling (Coello et al., 2011). SnRK2s are core components of the ABA‐dependent signaling pathway and operate within the PYL‐PP2C‐SnRK2 module, which underpins plant responses to abiotic stress (Takahashi et al., 2020). Extensive evidence shows that SnRK2s participate in plant responses to salinity, drought, and cold stress. In Arabidopsis thaliana, SnRK2.4 and SnRK2.10 regulate reactive oxygen species (ROS) homeostasis under salt stress; SnRK2.4 controls hydrogen peroxide levels, peroxidase activity, and ascorbic acid content, whereas SnRK2.10 is rapidly activated by salt and enhances tolerance by sustaining photosynthesis and limiting oxidative damage (Mazur et al., 2021; Szymanska et al., 2019). In Solanum nigrum, PtrSnRK2.4 enhances drought tolerance by promoting putrescine biosynthesis (Song et al., 2022). In rice, OsSAPK8 is strongly induced by drought, and its mutant shows lower drought tolerance at the vegetative stage and decreases yield (Zhong et al., 2020). Under low‐temperature conditions, OsSAPK8 mutants exhibit retarded growth, while overexpression of TaSnRK2.3 in Arabidopsis or transfer of AcSnRK2.11 into tobacco significantly improves cold tolerance (Tian et al., 2013; Xiang et al., 2020; Zhong et al., 2020).

Nitrogen uptake and transport in plants are mediated primarily by nitrate transporters (NRTs) and ammonium transporters (AMTs). The high‐affinity nitrate transporter NRT2 plays a key role in plant adaptation to low N conditions. Arabidopsis contains seven NRT2 members, five of which function in nitrate uptake across different tissues (Lv et al., 2022). Among them, NRT2.4 is localized to the plasma membrane of lateral root epidermal cells and stem tissues near the phloem, where it contributes to root nitrate uptake and long‐distance nitrate transport (Kiba et al., 2012). Under N deficiency, induction and plasma‐membrane localization of NRT2.5 are essential for adult plant growth and for efficient nitrate uptake and allocation to the phloem (Lezhneva et al., 2014). In addition, NRT2.5 and NRT2.6 jointly respond to rhizobium infection and counteract the suppression of lateral root development caused by high N availability (Dechorgnat et al., 2012). Dof1.7 regulates NRT2 expression in an N‐dependent manner: under N‐sufficient conditions, it directly activates NRT2.5, whereas under N deficiency it indirectly activates NRT2.1 and NRT2.4 by repressing NIGT1 expression (Zhuo et al., 2024). Following root nitrate uptake, NO3 − is reduced to NH4 + in plant cells. Ammonium transport is primarily mediated by AMT family proteins, and their expression is dynamically regulated by plant nitrogen status (Bajgain et al., 2018). For instance, exogenous nitrate supply strongly represses AMT1.2 expression in multiple plant species (Gonçalves et al., 2020). Transcriptional regulation constitutes a major layer of control for AMT gene expression, involving a diverse array of transcription factors belonging to different families. For example, the conserved bZIP transcription factor HY5 negatively regulates AtAMT1.2 in Arabidopsis and OsAMT1.1 in rice, while the rice DOF‐type transcription factor OsDOF18 exerts positive regulatory effects on OsAMT1.3, OsAMT2.1, and OsAMT4.1 (Huang et al., 2015; Wu et al., 2017).

Apple is an economically important fruit tree whose productivity is strongly affected by environmental stress. SnRKs are central regulators integrating nutrient metabolism and stress‐response signaling. Although SnRKs have been linked to N utilization (Lu et al., 2015), their role in apple responses to N deficiency remains unclear. We previously identified 12 SnRK2s in the apple genome and demonstrated that SnRK2.10 enhances drought and salt tolerance (Shao et al., 2019; Ye et al., 2022). In this study, we investigated the role of SnRK2.10 in low N stress responses and NUE in apple plants. We generated multiple transgenic apple lines targeting SnRK2.10 and two N transporter genes, NRT2.5 and AMT1.1. Our results show that SnRK2.10 positively regulates plant growth and NUE under low N conditions. SnRK2.10 directly interacts with NRT2.5 and AMT1.1, which may contribute to the enhanced N uptake of the apple plants during N deficiency. Collectively, our findings identify a SnRK2.10‐NRT2.5/AMT1.1 regulatory module that enhances N acquisition under low N stress, providing a theoretical basis and genetic resources for improving NUE and stress tolerance in apple breeding and production.

RESULTS

SnRK2.10 positively regulates the low N response and NUE in apple plants

SnRK2.10 was localized in both the cytoplasm and nucleus (Figure 1B), and its expression was induced by low N stress. Expression increased nearly 4‐fold at 4 days of low N treatment in leaves and nearly 7.5‐fold at 5 days of low N treatment in roots (Figure 1A). In addition, strong SnRK2.10 signals were detected in vascular tissues, including xylem and phloem, suggesting a potential role in N uptake in apple plants (Figure 1C). To verify the role of SnRK2.10 in regulating low N stress responses and NUE in apple plants, we used two previously generated transgenic apple lines overexpressing SnRK2.10 (OE1 and OE2; Figure 2; Shao et al., 2019). Under control conditions, no significant differences in growth phenotypes were observed between OE and WT plants. However, after 30 days of low N stress, the aerial parts of OE plants were similar to those of WT plants, whereas OE plants developed more extensive root systems than WT plants (Figure 2A). Under low N stress, OE plants exhibited higher Pn values than WT plants (Figure 2B), and both root dry weight and root activity (DHA) were also higher in OE plants (Figure 2C,D). Measurement of N content showed that OE plants had higher N content than WT plants (Figure 2E). Consequently, NUE was significantly higher in OE plants than in WT plants under low N stress (Figure 2F). To further confirm the role of SnRK2.10 in low N stress responses, we generated transgenic apple plants with suppressed SnRK2.10 expression (Ri1, Ri2, and Ri3; Figure S1A,B). Under control conditions, Ri plants showed growth comparable to WT plants, while under low N stress, Ri plants were smaller than WT plants (Figure 2G). Moreover, all enhanced physiological traits observed in OE plants were reversed in Ri plants, including Pn, root dry weight, DHA, N content, and NUE, which were all lower in Ri plants than in WT plants (Figure 2H–L). Together, these results indicate that SnRK2.10 acts as a positive regulator of low N stress responses and NUE in apple plants.

Figure 1.

Figure 1

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Expression patterns and subcellular localization of SnRK2.10.

(A) The expression level of SnRK2.10 in the leaves and roots under low N stress.

(B) Subcellular localization of apple SnRK2.10. Scale bars = 100 μm; BF, bright field; GFP, green fluorescent protein.

(C) In situ hybridization of SnRK2.10 in roots, leaves, and stems of GL‐3 apple (Malus domestica) under control conditions (CK) and under low N conditions (low N). ep, epidermis; xy, xylem; ph, phloem; pi, pith; co, cortex; cu, cuticle; ue, upper epidermis; le, lower epidermis; pa, palisade mesophyll cells; sp, spongy mesophyll cells; vb, vascular bundle; ve, vein. Scale bar = 20 μm. Data are mean ± SD. According to one‐way ANOVA and Duncan multiple range test, different letters represent significant differences between different plants (P < 0.05). CK, plants without any treatment; low N, low N stress treatment (0.02 mM N).

Figure 2.

Figure 2

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SnRK2.10 overexpression enhanced the tolerance of apple plants to low‐N stress.

(A) Phenotypes of WT and SnRK2.10‐OE apple lines after 30 days of cultivation under control or low N conditions. Scale bar: 10 cm.

(B) Net photosynthesis (Pn) in WT and SnRK2.10‐OE apple lines.

(C) Root dry weight in WT and SnRK2.10‐OE apple lines.

(D) DHA in WT and SnRK2.10‐OE apple lines.

(E) N content in WT and SnRK2.10‐OE apple lines.

(F) NUE in WT and SnRK2.10‐OE apple lines.

(G) Phenotypes of WT and SnRK2.10‐Ri apple lines after 30 days of cultivation under control or low N conditions. Scale bar = 10 cm.

(H) Net photosynthesis (Pn) in WT and SnRK2.10‐Ri apple lines.

(I) Root dry weight in WT and SnRK2.10‐Ri apple lines.

(J) DHA in WT and SnRK2.10‐Ri apple lines.

(K) N content in WT and SnRK2.10‐Ri apple lines.

(L) NUE in WT and SnRK2.10‐Ri apple lines. Data are mean ± SD. According to one‐way ANOVA and Duncan multiple range test, different letters represent significant differences among all the collected data (P < 0.05). WT, wild type, here we used GL‐3 apple (Malus domestic), which was also used as explants in generating transgenic apple plants; OE1/2, SnRK2.10‐overexpression line 1/2; Ri1/2/3, SnRK2.10‐RNA interference line 1/2/3.

Effects of SnRK2.10 on the transcriptional and translational levels in apple plants under low N stress

To investigate the biological basis by which SnRK2.10 regulates the apple response to low N stress, RNA‐seq analysis was first performed using OE (OE2) and WT plants. Using a 1.5‐fold change threshold, 4966 DEGs were identified in the OE/WT comparison (Figure S2B), including 1761 up‐regulated and 3205 down‐regulated genes (Figure S2A). Clustering analysis showed that many DEGs were associated with N nutrition. Specifically, down‐regulated DEGs in the OE/WT group were mainly enriched in RNA regulation, cell wall organization, central amino acid metabolism, and N transport (Figure S2C, down group). In contrast, up‐regulated DEGs were mainly enriched in RNA regulation, hormone metabolism, N metabolism, and DNA synthesis (Figure S2C, up group). These results suggest that the major transcriptional differences between OE and WT plants occur in RNA regulation and N nutrition. In addition, proteomic analysis identified 857 DEPs in the OE/WT comparison (Figure S3B), including 281 up‐regulated and 576 down‐regulated proteins, using a 1.2‐fold threshold (Figure S3A). Clustering of DEPs showed that many were also associated with N nutrition, particularly N transport (Figure S3C). A total of 17 N‐transport‐related DEGs were identified in the RNA‐seq data, including 3 AMTs, 10 DEGs encoding NRTs, and 4 encoding nitrate antiporters, with most showing higher expression in OE plants than in WT (Figure 3A; Table S2). In contrast, only 6 N‐transport–related DEPs were detected, including 3 AMTs, 2 NRTs, and 1 nitrate antiporter (Figure 3B, Supplemental Table 2), all of which were upregulated in OE plants. Notably, AMT1.1 (MD13G1225400) and NRT2.5 (MD13G1096700) were identified in both the DEG and DEP datasets. We performed qPCR analysis to examine the expression levels of these two genes in SnRK2.10‐OE and WT plants. The results showed that both AMT1.1 and NRT2.5 were expressed much higher in OE lines than in WT, and this pattern was also maintained under low nitrogen stress (Figure 3C), consistent with the RNA‐seq data. These results suggested that NRT2.5 and AMT1.1 may be the direct targets of SnRK2.10.

Figure 3.

Figure 3

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Through multi‐omics analysis, the nitrate transporter NRT2.5 and the ammonium transporter AMT1.1 were screened.

(A) The relative quantitative expression levels of N metabolism‐related genes in the transcriptome.

(B) The relative quantitative expression levels of N metabolism‐related proteins in the proteome. The colors indicate the expression of each DEG (A) and DEP (B) as determined by the average peak response area with Z‐score normalization.

(C) Relative expression levels of AMT1.1 and NRT2.5 in WT and SnRK2.10‐overexpressing lines. Data are mean ± SD. According to one‐way ANOVA and Duncan multiple range test, different letters represent significant differences among all the collected data (P < 0.05). WT, wild type, here we used GL‐3 apple (Malus domestica), which was also used as explants in generating transgenic apple plants; OE1/2 in (C), SnRK2.10‐overexpression line 1/2; WT.1/2/3 and OE.1/2/3 in (A) and (B) represent three biological replicates.

SnRK2.10 interacts with the N transporters and promotes N uptake under low N stress

To further verify whether SnRK2.10 interacts with NRT2.5 and AMT1.1, Y2H assays were performed. Yeast cells co‐transformed with SnRK2.10 and NRT2.5 or AMT1.1 grew well on ‐Trp/−Leu/‐His/−Ade medium (Figure 4A). When SnRK2.10 and NRT2.5 or AMT1.1 were co‐expressed in tobacco leaves, strong luciferase signals were detected using an ultra‐sensitive imaging system (Figure 4B,C). In pull‐down assays, purified SnRK2.10‐GST was incubated with NRT2.5‐His or AMT1.1‐His, and protein interactions were confirmed after pull‐down (Figure 4D,E). Collectively, these results demonstrate that SnRK2.10 interacts with NRT2.5 and AMT1.1, suggesting that SnRK2.10 may promote NUE by enhancing N uptake in apple plants.

Figure 4.

Figure 4

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SnRK2.10 interacts directly with NRT2.5 and AMT1.1.

(A) A Y2H assay indicates the interaction of SnRK2.10 and NRT2.5 or AMT1.1.

(B) A Split‐LUC assay indicates the interaction of SnRK2.10 and NRT2.5.

(C) A Split‐LUC assay indicates the interaction of SnRK2.10 and AMT1.1.

(D) A pull‐down assay indicates the interaction of SnRK2.10 and NRT2.5.

(E) A pull‐down assay indicates the interaction of SnRK2.10 and AMT1.1. Split‐LUC, split luciferase complementation assay; Y2H; yeast two‐hybrid.

We used NMT to measure the ion flow rates of NO3 − and NH4 + in the roots of WT and SnRK2.10 transgenic apple plants. The results showed that under control conditions, there was no significant difference in nitrate ion flow rates among the different plant lines. Under low N stress, nitrate flow rates significantly decreased in all plants. The nitrate flow rate in the OE plants was significantly higher than that in the WT plants, whereas the nitrate flow rate in the Ri plants was significantly lower than that in the WT plants (Figure 5A,B). Furthermore, under control conditions, there was no significant difference in NH4 + excretion rates among the different lines. However, under low N treatment, NH4 + absorption increased in all plants. The NH4 + absorption rate in the OE lines was significantly higher than that in the WT plants, whereas the rate in the Ri lines was significantly lower than that in the WT plants (Figure 5C,D). Although the low N conditions repressed net NO3 − influx, its absolute values were markedly greater than net NH4 + influx, suggesting that SnRK2.10 appears to preferentially regulate NRT2.5 in apple. These results suggested that SnRK2.10 promotes NUE in apple plants under low N stress, probably by regulating N uptake.

Figure 5.

Figure 5

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Effects of NO3 − flux and NH4 + flux in each line.

(A, B) Effects of low N stress on net NO3 − flux in apple plants with different expression levels of SnRK2.10.

(C, D) Effects of low N stress on net NH4 + flux in apple plants with different expression levels of SnRK2.10. The positive or negative sign of the flow rate value represents the direction of ion transport, with the positive as efflux and the negative as influx. Data are mean ± SD. According to one‐way ANOVA and Duncan multiple range test, different letters represent significant differences among different lines under control and low N stress, respectively (P < 0.05). WT, wild type, here we used GL‐3 apple (Malus domestic), which was also used as explants in generating transgenic apple plants; OE1/2, SnRK2.10‐overexpression line 1/2; Ri1/2/3, SnRK2.10‐RNA interference line 1/2/3.

NRT2.5 positively regulates the NUE of apple plants under low N stress

The expression of NRT2.5 in leaves increased 2.5‐fold after 4 days of low N treatment, while its expression in roots increased 5‐fold after 5 days of low N treatment (Figure S4A). In situ hybridization showed strong NRT2.5 signals in vascular tissues, including the xylem and phloem (Figure S4C), similar to those observed for SnRK2.10. This protein was mainly localized in the cytoplasm (Figure S4B). To investigate the role of NRT2.5 in the low N stress response of apple plants, we generated transgenic lines overexpressing NRT2.5 (OE1, OE2, and OE3; Figure S1C,D) and lines with suppressed NRT2.5 expression (Ri1, Ri2, and Ri3; Figure S1E,F). Under control conditions, no significant differences in growth phenotypes were observed between transgenic and WT plants. However, after 30 days of low N stress, OE plants showed milder leaf chlorosis, whereas Ri plants exhibited more severe leaf chlorosis compared with WT plants (Figure 6A,B). Although Pn decreased in all plants under low N stress, Pn values were significantly higher in OE lines than in WT plants and were lowest in Ri lines (Figure 6D). Under control conditions, chlorophyll fluorescence and Fv/Fm values did not differ between WT and transgenic plants. Under low N stress, the chlorophyll fluorescence was stronger in OE plants than in WT plants, and weakest in Ri plants (Figure 6C). Correspondingly, Fv/Fm values significantly decreased in all lines, but remained highest in OE plants, intermediate in WT plants, and lowest in Ri plants (Figure 6E). Additionally, OE plants developed stronger root systems, whereas Ri plants exhibited weaker root systems compared with WT plants (Figure 6A). Furthermore, under low N stress, root dry matter accumulation was higher in OE plants and lower in Ri plants relative to WT plants (Figure 6F). Root activity, assessed by DHA, decreased in all lines under low N stress. However, OE plants maintained the highest DHA values, Ri plants the lowest, and WT plants intermediate values (Figure 6G).

Figure 6.

Figure 6

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NRT2.5 overexpression enhanced the tolerance of apple plants to low N stress.

(A, B) Phenotypes of WT, NRT2.5‐OE and NRT2.5‐Ri apple lines after 30 days of cultivation under control or low N conditions. Scale bar = 10 cm.

(C) Chlorophyll fluorescence images.

(D) Net photosynthesis (Pn).

(E) Fv/Fm ratio.

(F) Root dry weight.

(G) Root activity DHA.

(H, I) Effects of low N stress on net NO3 − flux in different lines.

(J, K) Effects of low N stress on net NH4 + flux in different lines.

(L) N content.

(M) NUE. Data are mean ± SD. According to one‐way ANOVA and Duncan multiple range test, different letters in I and K represent significant differences among different lines under control and low N stress, respectively, and in others represent significant differences among all the collected data (P < 0.05). WT, wild type, here we used GL‐3 apple (Malus domestic), which was also used as explants in generating transgenic apple plants; OE1/2/3, NRT2.5‐overexpression line 1/2/3; Ri1/2/3, NRT2.5‐RNA interference line 1/2/3.

As the nitrate transporter, we also used NMT to measure NO3 − and NH4 + fluxes in WT and NRT2.5 transgenic apple roots. Under low N stress, the NO3 − influx was significantly higher in OE plants than in WT plants, and lowest in Ri plants (Figure 6H,I). A similar trend was observed for the NH4 + flux under low N stress (Figure 6J,K). The difference in the net influx made the total N content significantly higher in NRT2.5‐OE plants than in WT, and lower in NRT2.5‐Ri lines compared with WT plants (Figure 6L). The low N treatment significantly increased NUE in all lines. However, the NUE of OE1, OE2, and OE3 plants was 1.28‐, 1.19‐, and 1.41‐fold higher than that in WT plants, respectively, whereas NUE in Ri1, Ri2, and Ri3 plants was reduced to 0.57‐, 0.61‐, and 0.85‐fold of WT levels (Figure 6M). These results indicate that NRT2.5 positively regulates NUE in apple plants under low N stress.

AMT1.1 positively regulates the NUE of apple plants under low N stress

AMT1.1 was also localized in the cytoplasm (Figure S5B), and its expression level in leaves increased by approximately 2.5‐fold at 3 days of low N treatment, while in roots it increased by approximately 6‐fold at 5 days of low N treatment (Figure S5A). To better understand the role of AMT1.1 under low N stress, we generated three transgenic apple lines overexpressing AMT1.1 (OE1, −2, and −3, Figure S1G,H). AMT1.1 overexpression did not affect apple plant growth; however, under low N stress, leaf yellowing and reddening were less severe in OE plants than in WT plants, and OE plants developed stronger root systems than WT plants (Figure 7A,B). Subsequently, we measured the chlorophyll content of all lines and found that it decreased in all plants under low N stress, whereas all OE lines maintained significantly higher chlorophyll content than WT plants (Figure 7D). Under low N stress, the Pn values of OE lines were significantly higher than those of WT lines (Figure 7E). Additionally, the Fv/Fm values of all plants decreased significantly under low N stress, but the Fv/Fm values of OE plants remained higher than those of WT plants (Figure 7F), and a similar trend was observed in the chlorophyll fluorescence images (Figure 7C). The AMT1.1‐OE plants also exhibited stronger root systems, similar to those of NRT2.5‐OE plants. Accordingly, root length and root dry weight were significantly greater in AMT1.1‐OE lines than in WT plants (Figure 7G,H), and root activity (indicated by DHA value) was also higher in AMT1.1‐OE lines than in WT plants under low N stress (Figure 7I).

Figure 7.

Figure 7

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AMT1.1 overexpression enhanced the tolerance of apple plants to low N stress.

(A, B) Phenotypes of WT and AMT1.1‐OE apple lines after 30 days of cultivation under control or low N conditions. Scale bar = 10 cm.

(C) Chlorophyll fluorescence images.

(D) Total chlorophyll.

(E) Net photosynthesis (Pn).

(F) Fv/Fm ratio.

(G) Root length.

(H) Root dry weight.

(I) DHA.

(J, K) Effects of low N stress on net NO3 − flux in different lines.

(L, M) Effects of low N stress on net NH4 + flux in different lines.

(N) N content.

(O) NUE. Data are mean ± SD. According to one‐way ANOVA and Duncan's multiple range test, different letters in K and M represent significant differences among different lines under control and low N stress, respectively, and in others represent significant differences among all the collected data (P < 0.05). WT, wild type, here, we used ‘GL‐3’ apple (Malus domestica), which was also used as explants in generating transgenic apple plants; OE1/2/3, AMT1.1‐overexpression line 1/2/3.

We further investigated the role of AMT1.1 in N uptake using NMT technology to measure NO3 − and NH4 + fluxes in the roots of each line. The results showed that under control conditions, there were no significant differences in NO3 − uptake rates among the lines. However, under low N conditions, the NO3 − uptake rates in OE plants were significantly higher than those in WT plants (Figure 7J,K). Additionally, under low N conditions, NH4 + uptake rates in OE plants were also significantly higher than those in WT plants (Figure 7L,M). The increased N uptake resulted in significantly higher total N content in AMT1.1‐OE lines compared with WT plants under low N stress (Figure 7N). When NUE was measured, the NUE of OE plants was significantly higher than that of WT plants, with OE1, OE2, and OE3 exhibiting NUE values that were 1.27‐, 1.35‐, and 1.37‐fold higher than those of WT plants under low N stress, respectively (Figure 7O). These results indicate that AMT1.1 is a positive regulator of NUE and the low N stress response in apple plants.

SnRK2.10 collaborates with the N transporters in regulating the NUE of apple plants under low N stress

To further determine the effect of the combined action of SnRK2.10 and the two N transporters, NRT2.5 and AMT1.1, on low N stress, we first suppressed the expression of NRT2.5 in SnRK2.10‐Ri plants via VIGS (Figure S6A). When exposed to low N stress, suppression of NRT2.5 further reduced the stress tolerance of SnRK2.10‐Ri plants; leaves of SnRK2.10‐Ri/NRT2.5‐Ri plants showed more severe chlorosis and redness than those of SnRK2.10‐Ri and WT plants (Figure 8A). Compared with SnRK2.10‐Ri plants, SnRK2.10‐Ri/NRT2.5‐Ri plants had lower Fv/Fm values, and chlorophyll fluorescence imaging showed a similar trend (Figure 8C,D). In addition, root growth of SnRK2.10‐Ri plants was further inhibited by suppression of NRT2.5 (Figure 8B, SnRK2.10‐Ri/NRT2.5‐Ri vs. SnRK2.10‐Ri). DHA values were significantly lower in SnRK2.10‐Ri/NRT2.5‐Ri plants than in SnRK2.10‐Ri plants (Figure 8E). We also measured the flow rates of NO3 − and NH4 + ions in each line and found that both their absorption rates in SnRK2.10‐Ri/NRT2.5‐Ri plants were lower than those in SnRK2.10‐Ri plants under low N stress (Figure 8F–I). The total N content in SnRK2.10‐Ri/NRT2.5‐Ri plants was significantly lower than that in SnRK2.10‐Ri plants (Figure 8J), and the NUE of SnRK2.10‐Ri/NRT2.5‐Ri plants was significantly lower than that of SnRK2.10‐Ri plants under low N stress (Figure 8K).

Figure 8.

Figure 8

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SnRK2.10 and NRT2.5 cooperatively enhanced the tolerance of apple plants to low nitrogen stress.

(A) Phenotypes of SnRK2.10‐Ri apple plants with silenced NRT2.5 mediated by transient transformation under low N stress. Scale bar = 10 cm.

(B) Root system architecture scan diagram.

(C) Chlorophyll fluorescence images.

(D) Fv/Fm ratio.

(E) DHA.

(F, G) Effects of low N stress on net NO3 − flux in different lines.

(H, I) Effects of low N stress on net NH4 + flux in different lines.

(J) N content.

(K) NUE. Data are mean ± SD. According to one‐way ANOVA and Duncan multiple range test, different letters in G and I represent significant differences among different lines under control and low N stress, respectively, and in others represent significant differences among all the collected data (P < 0.05).

We then generated SnRK2.10‐OE/AMT1.1‐OE plants by transiently overexpressing SnRK2.10 in AMT1.1‐OE plants (Figure S6B). When exposed to low N stress, overexpressing both SnRK2.10 and AMT1.1 further enhanced the stress tolerance of apple plants than overexpressing AMT1.1 alone; leaves of SnRK2.10‐OE/AMT1.1‐OE plants showed less chlorosis and redness than those of AMT1.1‐OE and WT plants (Figure 9A). Compared with AMT1.1‐OE plants, SnRK2.10‐OE/AMT1.1‐OE plants had higher Fv/Fm values, and chlorophyll fluorescence imaging showed a similar trend under low N stress (Figure 9C,D). In addition, root scanning showed that, under low N stress, co‐overexpression of SnRK2.10 and AMT1.1 further enhanced root growth of apple plants compared with AMT1.1‐OE and WT plants (Figure 9B). DHA measurements revealed that SnRK2.10‐OE/AMT1.1‐OE plants had significantly enhanced root activity (Figure 9E). We also measured the flow rates of NO3 − and NH4 + ions in each line and found that both their absorption rates in SnRK2.10‐OE/AMT1.1‐OE plants were higher than those in AMT1.1‐OE plants (Figure 9F–I). The total N content in SnRK2.10‐OE/AMT1.1‐OE plants was significantly higher than that in AMT1.1‐OE plants (Figure 9J), and the NUE of SnRK2.10‐OE/AMT1.1‐OE plants was also significantly higher than that of AMT1.1‐OE plants under low N stress (Figure 9K).

Figure 9.

Figure 9

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SnRK2.10 and AMT1.1 cooperatively enhanced the tolerance of apple plants to low nitrogen stress.

(A) Phenotypes of AMT1.1‐OE apple plants with overexpressed SnRK2.10 mediated by transient transformation under low N stress. Scale bar = 10 cm.

(B) Root system architecture scan diagram.

(C) Chlorophyll fluorescence images.

(D) Fv/Fm ratio.

(E) DHA.

(F, G) Effects of low N stress on net NO3 − flux in different lines.

(H, I) Effects of low N stress on net NH4 + flux in different lines.

(J) N content.

(K) NUE. Data are mean ± SD. According to one‐way ANOVA and Duncan multiple range test, different letters in G and I represent significant differences among different lines under control and low N stress, respectively, and in others represent significant differences among all the collected data (P < 0.05).

Taken together, these results suggest that the interaction between SnRK2.10 and the N transporters (NRT2.5 and AMT1.1) functions effectively in apple plants under low N stress. They collaboratively promoted NUE under low N stress, probably by enhancing N transport.

DISCUSSION

Nitrogen is an essential macronutrient that plays a critical role in plant growth and development. N deficiency can reduce seed yield and chlorophyll content, cause leaf chlorosis, accelerate senescence, and impair overall plant growth, resulting in yield loss (Fu et al., 2022). The Loess Plateau is the largest apple‐producing region in China, the low NUE of apple varieties severely limits yield and fruit quality here. Functional genomic studies have revealed a number of genes involved in low N responses or the NUE regulation in apple plants. For instance, the transcription factor MdNLP7 promotes N uptake and assimilation by activating the expression of MdNIA2 and MdNRT1.1 (Feng et al., 2022). The miR164‐MhNAC1 module plays a role in the N uptake of apple rootstocks, as well as in the regulation of citric acid secretion in roots (Wang, Xu, et al., 2024). The SnRKs members play important roles in enhancing plant tolerance to various stresses. For example, CiPawSnRK2.1 deletion significantly reduces salt tolerance in walnut seedlings (Wang, Zhou, et al., 2024), AcSnRK2.4 positively regulates heat stress responses in kiwifruit (Wang et al., 2025), and TaSnRK2.4‐B negatively regulates pathogen resistance through an ABA‐dependent pathway (Jiang et al., 2022). Our previous studies demonstrated that overexpression of SnRK2.10 in Arabidopsis and apple enhances tolerance to drought and salt stress (Shao et al., 2019; Ye et al., 2022). Here, we further investigated the role of SnRK2.10 in regulating NUE in apple plants. The low N treatment induced SnRK2.10 expression in both roots and leaves (Figure 1). Overexpression of SnRK2.10 significantly enhanced low N stress tolerance and NUE (Figure 2), whereas its suppression reduced low N tolerance and NUE in apple plants (Figure 2), indicating that SnRK2.10 plays a positive role in regulating NUE under low N stress.

Under N‐deficient conditions, plants develop deeper and more extensive root systems to enhance N uptake, representing an important adaptive strategy (Aggarwal et al., 2025; Ju et al., 2015; Rasmussen et al., 2015; Smoczynska et al., 2022). OsNAR2.2 enhances rice resistance to N starvation by promoting root growth and N uptake (Xu et al., 2020). PpNrt2.1 and PpNrt2.2 are specifically expressed in the roots of peach trees and are involved in the absorption of nitrate in the roots of peach trees (Nakamura et al., 2007). In this study, we found that plants overexpressing SnRK2.10 exhibited more developed root systems and higher root vitality under low N stress (Figure 2), suggesting that SnRK2.10 may also have enhanced N uptake efficiency. The integrated multi‐omics analysis indicated that SnRK2.10 may regulate N transport in response to low N (Figure 3). Further analysis confirmed that SnRK2.10 directly interacts with two N transport proteins, NRT2.5 (nitrate transporter) and AMT1.1 (ammonium transporter) (Figure 4). N uptake capacity is closely associated with NUE, and enhancing N uptake is a key strategy for improving crop NUE. For instance, OsNRT1.1A overexpression in rice significantly increased NUE and yield and also shortened the maturation period (Wang et al., 2018). Polymorphisms in OsNRT1.1B may lead to functional differences in NUE improvement in rice (Siangliw et al., 2024). Coordinated regulation of BnNRT1.5 and BnNRT1.8 promotes nitrate distribution to aboveground tissues in rapeseed‐type oil crops and improves NUE (Han et al., 2016). The ammonium transporter genes BcAMT1s differentially regulate the absorption and accumulation of NH4 + and NO3 −, thereby influencing plant NUE (Straub et al., 2017). Although NRT2.5 and AMT1.1 are annotated as N transporters, their biological roles in apple under low N stress remain unclear. Therefore, we generated overexpression and silencing lines for these two genes for functional analysis. Overexpression of NRT2.5 enhanced plant tolerance to low N stress, whereas silencing of NRT2.5 reduced low N stress tolerance (Figure 6). In addition, overexpression of AMT1.1 also enhanced low N stress tolerance (Figure 7), consistent with previous findings. Ion flux analysis showed that NRT2.5 expression significantly affects NO3 − uptake and transport (Figure 6), while AMT1.1 overexpression promotes NH4 + influx under low N stress (Figure 7). These results are consistent with the known functions of NRTs and AMTs, indicating that NRT2.5 and AMT1.1 improve NUE in transgenic plants by regulating NO3 − and NH4 + uptake.

SnRK2.10 interacted with both NRT2.5 and AMT1.1, suggesting that it may also affect N uptake to regulate NUE in apple plants. Ion flux analysis in SnRK2.10 transgenic plants showed that, under low N stress conditions, plants overexpressing SnRK2.10 exhibited higher NO3 − and NH4 + absorption than WT plants (Figure 5), preliminarily confirming that SnRK2.10 has a regulatory role in N uptake. As a member of the SnRK2 protein kinase family, SnRK2.10 is likely to regulate the activity of its interacting proteins through phosphorylation. The protein kinases, such as CIPK23, regulate AMTs and thereby influence NH4 + and NO3 − uptake and accumulation (Straub et al., 2017). Similarly, the protein kinase OsACTPK1 mediates phosphorylation of OsAMT1 to prevent excessive ammonium accumulation in plants (Beier et al., 2018). Therefore, we speculate that SnRK2.10 may also phosphorylate NRT2.5 and AMT1.1 to alter their activity, stability, or subcellular localization, thereby optimizing transport function and ultimately enhancing plant NUE. The transport activity of NRTs is closely associated with phosphorylation at specific sites, enabling plants to adapt to different N conditions. When external nitrate levels decrease, phosphorylation of T101 converts NRT1.1 from a low‐affinity to a high‐affinity transporter, thereby regulating auxin flux and nitrate signal transduction during root growth (Bouguyon et al., 2016; Zhang et al., 2019). In contrast, phosphorylation of S585 markedly weakens its nitrate transport capacity (Su et al., 2021). Phosphorylation of T98 in NRT1.4 similarly reduces its transport activity (de los Ríos et al., 2021). When external nitrate levels increase, phosphorylation of S501 inhibits the transport activity of NRT2.1 and reduces high‐affinity nitrate uptake by roots (Jacquot et al., 2020). The transport activity of AMTs is also tightly regulated by phosphorylation at different sites. Mutation of T460 of AtAMT1.1 in Arabidopsis thaliana abolishes ammonium uptake, and similar effects can be observed for corresponding mutations in AtAMT1.2 (T472) and AtAMT1.3 (T464). These findings indicate that phosphorylation may be a conserved way of regulating the activity of these transporters (Neuhäuser et al., 2007; Wu et al., 2019). Constrained by technical challenges in phosphorylation experiments, we could not fully confirm the phosphorylation of NRT2.5 (or AMT1.1) by SnRK2.10. However, we constructed two kinds of co‐transgenic apple plants and analyzed their responses to low N stress, as well as their NUE, thus verifying the biological function of the interaction between SnRK2.10 and the two transporters (Figure S6). Under low N stress, overexpressing both SnRK2.10 and N transporter gene (AMT1.1) further enhanced the stress tolerance of apple plants compared with overexpressing AMT1.1 alone (Figure 9); while suppressing the expression of the N transporter gene (NRT2.5) in SnRK2.10‐OE plants significantly attenuated the low N stress tolerance of apple plants (Figure 8). These suggested that SnRK2.10 positively regulates plant growth and NUE under low N stress, probably partially by the direct interaction with key N transporters.

Naturally, the present work is not without any limitations. First, the results relied on OE and Ri plants, although their phenotypes were consistent with their tolerance, they were not enough to resolve whether the observed effects are direct or mediated through broader changes in plant metabolism or growth. This is also a common issue that can be observed in current pomology studies (Rahmati et al., 2018; Kumar et al., 2021; Habibi et al., 2023; Liu et al., 2023). Given that the molecular changes typically occur earlier than physiological responses, we preferred to believe that the interaction between SnRK2.10 and NRT2.5 (or AMT1.1) helps to enhance N uptake, and ultimately improves apple growth under low N stress. Second, we overemphasized the consistency in measured indicators and ignored the unique functional characteristics of each gene, leading to some overlap in the functional characterization of SnRK2.10, NRT2.5, and AMT1.1. SnRK2.10 is a kinase, while the other two are N transporters, there should be different manner when they execute their biological function. Third, the synergistic effects of SnRK2.10 and NRT2.5 (or AMT1.1) have been proposed but have not been fully elucidated, since we only employed two kinds of co‐transgenic apple materials. The major technical challenge is that stable genetic transformation of woody species such as apple is highly time‐consuming and technically demanding. This limitation prevented us from more precisely separating the individual effects of SnRK2.10, NRT2.5, and AMT1.1 from their combined effects. The last one, the controlled conditions setting in the present work, allowed us to clearly evaluate the function of SnRK2.10 and its interaction with NRT2.5/AMT1.1 in regulating N uptake and NUE, they cannot fully represent field settings or more complex soil environments. In particular, soil nitrogen availability, microbial activity, root‐soil interactions, seasonal environmental fluctuations, and orchard management practices may all influence N uptake and NUE of apple plants (Cao et al., 2019; Liang et al., 2025; Zhao et al., 2020). Collectively, these shortcomings offer valuable insights for our subsequent work, helping us achieve more robust research work.

CONCLUSIONS

In summary, this study demonstrated that SnRK2.10 was induced by low N stress in both roots and leaves, and that it positively regulated NUE and low N adaptive responses in apple plants. The omics analyses revealed that SnRK2.10 modulated N metabolism pathways and suggested that N transporters contribute to its positive regulatory function. Further assays confirmed that SnRK2.10 interacts with the N transporters NRT2.5 and AMT1.1. Both transporters acted as positive regulators of N uptake and NUE in apple plants. In addition, NO3 − and NH4 + fluxes in roots of SnRK2.10 transgenic apple lines were also significantly changed under low N stress (Figure 10). Overall, these results indicate that SnRK2.10 positively regulates NUE in apple plants, most likely via functional cooperation with NRT2.5 and AMT1.1. Additional genetic evidence is required to fully clarify their individual and combined effects. Nevertheless, SnRK2.10 is a promising target for genetic engineering to improve NUE in apple.

Figure 10.

Figure 10

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A working model demonstrating the role of SnRK2.10 in the response of apple to low N stress.

Low N stress induces the expression of SnRK2.10, NRT2.5, and AMT1.1. SnRK2.10 can interact with both NRT2.5 and AMT1.1, and this interaction probably affects the transporter activity of NRT2.5 and AMT1.1. The transport rates of both NO3 − and NH4 + in the SnRK2.10‐OE plants are enhanced under low N stress, leading to continued growth of the OE plants. Collectively, SnRK2.10 positively regulates the tolerance of apple plants to low N stress, probably via synergistic function with the N transporter NRT2.5 and AMT1.1. Note: the drawing elements in the model are generated with the help of AI (Doubao). For instance, the barrel‐shaped transporters, the spheres with different colors, the rectangles with different gene names, and the irregular ellipse we used to mark the SnRK2.10 protein.

MATERIALS AND METHODS

Plant materials and treatments

‘GL‐3’, a cultivar derived from ‘Royal Gala’ apple (Malus domestica), was used as the wild‐type (WT) plant in genetic transformation, transient expression, and virus‐induced gene silencing (VIGS). Nicotiana benthamiana served as a heterologous expression system.

For the low N stress treatments, GL‐3 and transgenic apple plants were grown on rooting medium for 35 days and then transplanted into plastic pots (8 × 8 cm) containing a substrate of vermiculite:perlite:soil (3:1:1, v/v/v). Plants were maintained in a controlled growth chamber under cool white light (16 h light/8 h dark, 8000 lx). After 1 month, uniformly growing apple plants were selected for hydroponic low‐N treatment, following the previously described methods (Jia et al., 2022; Li et al., 2022). After a 7‐days pre‐culture, both WT and transgenic plants were randomly assigned to 0.02 mM N (Low N treatment) or 6 mM N (Control) for 30 days.

Genetic transformation of apple plants

The SnRK2.10 overexpression lines (OE1 and OE2) were previously generated by Shao et al. (2019). When generating transgenic apple lines overexpressing NRT2.5 (or AMT1.1), the full coding sequence (CDS) of NRT2.5 (or AMT1.1) was cloned into pCambia2300. Selected SnRK2.10 and NRT2.5 fragments were cloned into the phellsgate vector to generate RNAi constructs. The recombinant plasmids were introduced into Agrobacterium strain GV3101. Stable and transient transformations were performed following established protocols (Liu et al., 2025; Zhang et al., 2024). All the primers were listed in Table S1.

In situ hybridization experiment

The in situ hybridization of SnRK2.10 and NRT2.5 was performed in apple tissues with minor modifications according to the previously described methods (Zhang et al., 2023). Roots, stems, and leaves were harvested and immediately fixed in FAA solution. Samples were decolorized, paraffin‐embedded, and sectioned. Sections were incubated at 37°C for 8–12 h, deparaffinized with xylene, and rehydrated using graded ethanol and DEPC‐treated water. Finally, the sections were then digested with 1 μg/mL proteinase K at 37°C, hybridized with probes at 42°C overnight, rinsed with washing buffer at 23°C and 65°C, and stored in TE buffer.

Quantitative PCR (qPCR) analysis

The total RNA was extracted using a Wolact Plant RNA Isolation Kit (Wolact, Hong Kong, China). The first‐strand cDNA was synthesized from 1 μg of the total RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). The qPCR assays were conducted using SYBR Green qPCR Kits (Takara, Tokyo, Japan) on an iQ5 Multicolor Detection System (Bio‐Rad Laboratories, Hercules, CA, USA), according to Zhang et al. (2023). All the primers were listed in Table S1.

Subcellular localization

The pCAMBIA2300‐GFP vector was digested with BamH I and Sal I, and the CDS of SnRK2.10, NRT2.5, and AMT1.1 were cloned by homologous recombination. Activated Agrobacterium cultures were resuspended to an OD600 of 0.6 and infiltrated into the leaves of Nicotiana benthamiana. Fluorescence signals were observed using a confocal laser scanning microscope (TCS‐SP8 SR; Leica, Germany).

Photosynthetic indicators

After 30 days of low N stress treatment, the chlorophyll content was determined following the previously described method (Ye et al., 2022). Briefly, 0.1 g of leaf tissue was homogenized in 4 mL of 80% acetone and incubated in darkness for 24 h. Absorbance was measured using a UV–Vis spectrophotometer (UV‐2600, Shimadzu, Japan). The net photosynthetic rate (Pn) was measured using a CIRAS‐3 portable photosynthesis system (Amesbury, MA, USA). Following 30 min of dark adaptation, chlorophyll fluorescence parameters were recorded using an IMAGING‐PAM fluorometer (WALZ, Nuremberg, Germany). Leaf fluorescence images were analyzed to evaluate photosystem damage under different treatments.

Measurements of growth parameters, N concentration, and stable isotope δ15N‐related indicators

At the end of the low N treatment, five plants per group were harvested for growth analysis. Root length was measured from the plant base to the tip of the main root. Root activity (DHA) was determined using the DHA detection kit (Suzhou Comin Biotechnology Co., Ltd, Suzhou, China). The root architecture was scanned using the SNAPSCAN 310 scanner, and the images were generated using the WinRhizo image analysis system.

For the N measurements, samples were fixed at 105°C for 30 min and dried at 65°C for 4 days. The root dry weight was then measured. The dried tissues (the whole plant, including roots, stems, and leaves) were ground into fine powder for N analysis. N concentration was determined as described previously (Jia et al., 2022). The stable isotope δ15N was measured using an elemental analyzer (Flash EA 1112HT, Thermo Fisher Scientific, USA) coupled with a stable isotope mass spectrometer (Finnigan Delta V Advantage, Thermo Fisher Scientific, USA).

Determination of flux of NO3 −/NH4 +

The net NO3 − /NH4 + flux was measured using non‐invasive micro‐test technology (NMT; Younger USA, LLC, Amherst, MA, USA). Flux measurements were performed approximately 20 μm from the root tip along the root axis. After 15 days of low N and control treatments, tender root tips were transferred to measurement solutions and equilibrated for 30 min. Ion fluxes were then recorded for 5 min and analyzed using imFluxes software (v2.0; Beijing Xuyue Technology Co., Ltd.). Three independent biological replicates were conducted, each comprising three randomly selected roots per line.

RNA‐seq and proteomic analyses

Under the control conditions, leaf samples were collected from SnRK2.10‐OE2 and WT for transcriptomic analysis by Wuhan Seqhealth Tech Co., Ltd. Raw sequencing data were aligned to the apple reference genome (GDDH13_v1.1_GDR) using Base Calling. Quality control, transcriptome assembly, and quantification were performed using fastp (v0.23.0). Differentially expressed genes (DEGs) were identified using DESeq2, with significance thresholds of adjusted P < 0.05 and |fold change| ≥1.5.

In addition, the leaf samples were collected for proteomic analysis by Beijing Tsingke Biotech Co., Ltd. Raw mass spectrometry data were processed using MaxQuant (v1.6.15.0), and MS/MS spectra were searched against the Blast_Malus_domestica_3750_PR_20210319.fasta database containing 42 478 entries. Differentially expressed proteins (DEPs) were identified using DESeq2, with significance thresholds of adjusted P < 0.05 and |fold change| ≥1.2.

Protein interaction assays

For yeast two‐hybrid (Y2H) assays, the pGBKT7 vector was digested with EcoR I and Sal I, and the NRT2.5 CDS and AMT1.1 CDS were inserted using homologous recombination, respectively. The pGADT7 vector was digested with BamH I and EcoR I, and the SnRK2.10 CDS was inserted into it. The resulting constructs were co‐transformed into the yeast strain Y2H‐Gold. Monoclonal colonies selected on SD/−Trp‐Leu medium were transferred to SD/−Trp‐Leu‐His‐Ade medium and incubated at 28°C for 3 days to assess yeast growth.

For the Split luciferase (Split‐LUC) complementation assays, the pCAMBIA1300‐n/cLUC vector was digested with Kpn I and Sal I. The NRT2.5 CDS and AMT1.1 CDS were cloned into pCAMBIA1300‐nLUC, respectively, and the SnRK2.10 CDS was cloned into pCAMBIA1300‐cLUC via homologous recombination. Recombinant plasmids were transformed into Agrobacterium tumefaciens GV3101, and bacterial suspensions were adjusted to an OD600 of 1.0–1.2. Different combinations were infiltrated into Nicotiana benthamiana leaves, and luciferase activity was detected 48–72 h after infiltration using an in vivo imaging system (PlantView100).

For the pull‐down assay, the SnRK2.10 CDS was cloned into pGEX4T‐1 with a GST tag, and the NRT2.5 CDS and AMT1.1 CDS were cloned into pET28a with a His tag, respectively. The recombinant plasmids were transformed into Escherichia coli BL21 (DE3), and protein expression was induced with IPTG at 4°C for 24 h. Proteins were purified according to the manufacturer's protocol (TianDiRenSheng, Smart‐Lifesciences, Jiangsu, China), and immunoblotting was performed using anti‐GST and anti‐His antibodies (Bioss, Beijing, China).

Statistical analysis

All data are presented as mean ± SD. Statistical analyses were performed using one‐way analysis of variance (ANOVA) in SPSS 27.0. Differences among data were evaluated using Duncan's multiple range test, with statistical significance set at P < 0.05, marked with different lowercase letters.

Author Contributions

FM and XG conceived the ideas; YY, ZZ, ZL, LL, XH and YD collected and analyzed the data; Ying Ye and Xiaoqing Gong wrote the manuscript; FM and XG revised the manuscript and provided financial support. All authors contributed critically to the drafts and gave final approval for publication.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Supporting information

Figure S1. Identification of transgenic apple plants. (A) Identification of the SnRK2.10 RNAi lines via PCR. (B) qPCR analysis of SnRK2.10 in the RNAi lines. (C) Identification of the NRT2.5 overexpression lines via PCR. (D) qPCR analysis of NRT2.5 in the overexpression lines. (E) Identification of the NRT2.5 RNAi lines via PCR. (F) qPCR analysis of NRT2.5 in the RNAi lines. (G) Identification of the AMT1.1 overexpression lines via PCR. (H) qPCR analysis of AMT1.1 in the overexpression lines. Data are mean ± SD. According to one‐way ANOVA and Duncan multiple range test, different letters represent significant differences between different plants (P < 0.05).

TPJ-127-0-s004.jpg (488.9KB, jpg)

Figure S2. Analysis of DEGs in WT and SnRK2.10 overexpression apple leaves. (A) Number of upregulated and downregulated differentially expressed genes (DEGs). (B) Cluster diagram of DEGs. (C) Mapman enrichment analyses between the WT and SnRK2.10‐OE. The colors in B indicate the expression of each DEG as determined by the average peak response area with Z‐score normalization. WT, wild type, here we used GL‐3 apple (Malus domestic), which was also used as explants in generating transgenic apple plants; OE, SnRK2.10‐overexpression line, here we used OE2.

TPJ-127-0-s006.jpg (1.2MB, jpg)

Figure S3. Analysis of DEPs in WT and SnRK2.10 overexpression apple leaves. (A) Number of upregulated and downregulated differentially expressed proteins (DEPs). (B) Cluster diagram of DEPs. (C) Mapman enrichment analyses between the WT and SnRK2.10‐OE. The colors in B indicate the expression of each DEP as determined by the average peak response area with Z‐score normalization. WT, wild type, here we used GL‐3 apple (Malus domestic), which was also used as explants in generating transgenic apple plants; OE, SnRK2.10‐overexpression line, here we used OE2.

TPJ-127-0-s007.jpg (1.2MB, jpg)

Figure S4. Expression patterns and subcellular localization of apple NRT2.5. (A) The expression level of NRT2.5 in the leaves and roots under low N stress. (B) Subcellular localization of apple NRT2.5. Scale bar = 100 μm; BF, bright field; GFP, green fluorescent protein. (C) In situ hybridization of NRT2.5 in roots, stems, and leaves of GL‐3 apple (Malus domestica) under control conditions (CK) and under low N conditions (low N). ep, epidermis; xy, xylem; ph, phloem; pi, pith; co, cortex; cu, cuticle; ue, upper epidermis; le, lower epidermis; pa, palisade mesophyll cells; sp, spongy mesophyll cells; vb, vascular bundle; ve, vein. Scale bar = 20 μm. Data are mean ± SD. According to one‐way ANOVA and Duncan multiple range test, different letters represent significant differences between data (P < 0.05).

TPJ-127-0-s003.jpg (853.6KB, jpg)

Figure S5. Expression patterns and subcellular localization of apple AMT1.1. (A) Subcellular localization of apple AMT1.1. Scale bar= 100 μm; BF, bright field; GFP, green fluorescent protein. (B) The expression level of AMT1.1 under low N stress. Data are mean ± SD. According to one‐way ANOVA and Duncan multiple range test, different letters represent significant differences between data (P < 0.05).

TPJ-127-0-s008.jpg (325.8KB, jpg)

Figure S6. Identification of transgenic apples. (A) qPCR identification of SnRK2.10 ‐ Ri apple plants with silenced NRT2.5. (B) qPCR identification of AMT1.1‐ OE apple plants with overexpressed SnRK2.10. Data are mean ± SD. According to one‐way ANOVA and Duncan multiple range test, different letters represent significant differences between data (P < 0.05).

TPJ-127-0-s002.jpg (141.3KB, jpg)

Table S1. The primers used in this study.

TPJ-127-0-s005.xlsx (11.3KB, xlsx)

Table S2. The detailed information of the 17 DEGs and 6 DEPs showed in Figure 3.

TPJ-127-0-s001.xlsx (12.9KB, xlsx)

Acknowledgements

The authors are grateful to Dr Zhihong Zhang from Shenyang Agricultural University for providing tissue‐cultured GL‐3 plants. We thank Miss Wenting Zhang (State Key Laboratory for Crop Stress Resistance and High‐Efficiency Production, Northwest A&F University, Yangling, China) for NMT experimental assistance. We also thank Yangyang Yuan from the horticultural science research center of the College of horticulture.

This work was supported by the National Natural Science Foundation of China (32472667, U24A20414), the Natural Science Basic Research Program of Shaanxi Province (2026JC‐QYCX‐024), the earmarked fund for the China Agricultural Research System (CARS‐27), and the Chinese Central Government Guidance for Local Science and Technology Development (2024ZY‐JCYJ‐02‐36).

Contributor Information

Fengwang Ma, Email: fwm64@nwsuaf.edu.cn.

Xiaoqing Gong, Email: gongxq0103@nwsuaf.edu.cn.

Data Availability Statement

The omics data are available for limited sharing upon reasonable request, as appropriate.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Identification of transgenic apple plants. (A) Identification of the SnRK2.10 RNAi lines via PCR. (B) qPCR analysis of SnRK2.10 in the RNAi lines. (C) Identification of the NRT2.5 overexpression lines via PCR. (D) qPCR analysis of NRT2.5 in the overexpression lines. (E) Identification of the NRT2.5 RNAi lines via PCR. (F) qPCR analysis of NRT2.5 in the RNAi lines. (G) Identification of the AMT1.1 overexpression lines via PCR. (H) qPCR analysis of AMT1.1 in the overexpression lines. Data are mean ± SD. According to one‐way ANOVA and Duncan multiple range test, different letters represent significant differences between different plants (P < 0.05).

TPJ-127-0-s004.jpg (488.9KB, jpg)

Figure S2. Analysis of DEGs in WT and SnRK2.10 overexpression apple leaves. (A) Number of upregulated and downregulated differentially expressed genes (DEGs). (B) Cluster diagram of DEGs. (C) Mapman enrichment analyses between the WT and SnRK2.10‐OE. The colors in B indicate the expression of each DEG as determined by the average peak response area with Z‐score normalization. WT, wild type, here we used GL‐3 apple (Malus domestic), which was also used as explants in generating transgenic apple plants; OE, SnRK2.10‐overexpression line, here we used OE2.

TPJ-127-0-s006.jpg (1.2MB, jpg)

Figure S3. Analysis of DEPs in WT and SnRK2.10 overexpression apple leaves. (A) Number of upregulated and downregulated differentially expressed proteins (DEPs). (B) Cluster diagram of DEPs. (C) Mapman enrichment analyses between the WT and SnRK2.10‐OE. The colors in B indicate the expression of each DEP as determined by the average peak response area with Z‐score normalization. WT, wild type, here we used GL‐3 apple (Malus domestic), which was also used as explants in generating transgenic apple plants; OE, SnRK2.10‐overexpression line, here we used OE2.

TPJ-127-0-s007.jpg (1.2MB, jpg)

Figure S4. Expression patterns and subcellular localization of apple NRT2.5. (A) The expression level of NRT2.5 in the leaves and roots under low N stress. (B) Subcellular localization of apple NRT2.5. Scale bar = 100 μm; BF, bright field; GFP, green fluorescent protein. (C) In situ hybridization of NRT2.5 in roots, stems, and leaves of GL‐3 apple (Malus domestica) under control conditions (CK) and under low N conditions (low N). ep, epidermis; xy, xylem; ph, phloem; pi, pith; co, cortex; cu, cuticle; ue, upper epidermis; le, lower epidermis; pa, palisade mesophyll cells; sp, spongy mesophyll cells; vb, vascular bundle; ve, vein. Scale bar = 20 μm. Data are mean ± SD. According to one‐way ANOVA and Duncan multiple range test, different letters represent significant differences between data (P < 0.05).

TPJ-127-0-s003.jpg (853.6KB, jpg)

Figure S5. Expression patterns and subcellular localization of apple AMT1.1. (A) Subcellular localization of apple AMT1.1. Scale bar= 100 μm; BF, bright field; GFP, green fluorescent protein. (B) The expression level of AMT1.1 under low N stress. Data are mean ± SD. According to one‐way ANOVA and Duncan multiple range test, different letters represent significant differences between data (P < 0.05).

TPJ-127-0-s008.jpg (325.8KB, jpg)

Figure S6. Identification of transgenic apples. (A) qPCR identification of SnRK2.10 ‐ Ri apple plants with silenced NRT2.5. (B) qPCR identification of AMT1.1‐ OE apple plants with overexpressed SnRK2.10. Data are mean ± SD. According to one‐way ANOVA and Duncan multiple range test, different letters represent significant differences between data (P < 0.05).

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Table S1. The primers used in this study.

TPJ-127-0-s005.xlsx (11.3KB, xlsx)

Table S2. The detailed information of the 17 DEGs and 6 DEPs showed in Figure 3.

TPJ-127-0-s001.xlsx (12.9KB, xlsx)

Data Availability Statement

The omics data are available for limited sharing upon reasonable request, as appropriate.

Articles from The Plant Journal are provided here courtesy of Wiley

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