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. 2026 Mar 16;19(1):e70215. doi: 10.1002/tpg2.70215

Genome‐wide Identification and expression analysis of TOR signaling pathway components in apple in response to low nitrogen

Dehai Liu 1, Siyuan Cheng 1, Yanghao Long 1, Qinyao Jin 1, Yujie Zhou 1, Fengwang Ma 1, Xuan Liu 1,✉, Xiaoqing Gong 1,✉
PMCID: PMC12993103  PMID: 41840890

Abstract

Target of rapamycin (TOR) is an evolutionarily conserved serine/threonine kinase that plays a central role in regulating biological growth, development, and stress responses in eukaryotes. However, the TOR signaling pathway has not been thoroughly studied in apple (Malus domestica). Here, through sequence alignment with homologous genes in Arabidopsis thaliana, 14 conserved members of the TOR signaling pathway, encoded by 28 sequences, were identified in the apple genome. A comprehensive analysis of these members was subsequently performed by integrating their structural features, phylogenetic relationships, and expression profiles under low‐nitrogen stress conditions. The results showed that the functional motifs of these members are highly conserved across species, while there are significant differences in the physicochemical properties of each member in apple. Subcellular localization predictions indicated that most members are likely localized to the nucleus; a few may reside in the cytoplasm or chloroplast. Quantitative PCR analysis showed that TOR pathway members are differentially regulated under low‐nitrogen stress, suggesting their potential involvement in nitrogen stress response. Furthermore, MdTORs were found to directly interact with several autophagy‐related (ATG) proteins in apple plants, in addition to its canonical target ATG13. Collectively, this study systematically characterizes the components of the TOR pathway in the apple genome, examines their expression dynamics under low nitrogen stress, and identifies novel interactions between TOR and ATGs. These research findings broaden our understanding of TOR‐regulated autophagic pathways, provide a valuable foundation for future studies into their regulatory mechanisms, and also provide data support for clarifying the responses of TOR signaling pathway members in apple to low‐nitrogen stress.

Core Ideas

  • A total of 14 members in the target of rapamycin (TOR) pathway were identified in the apple genome.

  • TOR signal members respond to low nitrogen stress.

  • MdTOR interacts with multiple MdATGs in addition to conserved MdATG13.

Plain Language Summary

Target of rapamycin (TOR) is an evolutionarily conserved serine/threonine kinase that plays an essential role in regulating biological growth, development, and stress responses in eukaryotes. Here, we identified all conserved members in the TOR pathway in the apple genome via analysis of their homologs in Arabidopsis. A total of 28 sequences encoding 14 typical members in the TOR pathway were identified in the apple genome. It was found that the gene expression pattern is induced by low‐nitrogen stress, and the expression of these genes reaches a peak on the third or fouth day. MdTORs can directly interact with several autophagy‐related protein (ATG) proteins in apple plants aside from its conserved target protein ATG13. Therefore, our results deepen the understanding of the mechanism by which TOR regulates autophagy and provide valuable data for future studies on the regulation of the TOR signaling pathway in apples under low‐nitrogen stress.

Abbreviations

ATG

autophagy‐related

CDS

coding sequence

Chr

chromosome

Expasy

expert protein analysis system

FAT

FRAP‐ATM‐TRRAP domain

FRB

FKBP12‐rapamycin‐binding domain

GDR

genome database for rosaceae

GFF3

general feature format version 3

HEAT

huntingtin, EF3, PP2A A subunit, TOR1

Ka/Ks

non‐synonymous substitution rate/synonymous substitution rate

MEME

multiple Em for Motif Elicitation

ML

maximum likelihood

NCBI

National Center for Biotechnology Information

qPCR

quantitative polymerase chain reaction

TOR

target of rapamycin

WGD

whole genome duplication

Y2H

yeast two‐hybrid

1. INTRODUCTION

Target of rapamycin (TOR), a conserved serine/threonine protein kinase widely present in eukaryotes, belongs to the PI3K kinase family and serves as a central regulator that responds to environmental signals and modulates cell growth and metabolism. In plants, only one protein complex formed by TOR kinase, namely, TORC1, has been identified so far, whereas the rapamycin‐insensitive TORC2 has not yet been detected in plants (Reinke et al., 2004). In plants, Growth factors, nutrients, energy, and stress are the major upstream signaling factors of TOR (Takahara & Maeda, 2013). After sensing upstream signals, TOR regulates a variety of cellular processes, including ribosome biogenesis, protein translation, lipid synthesis, autophagy, cell proliferation, survival, and cytoskeleton integration. It achieves this by two main mechanisms: modulating the transcription and translation of downstream genes or directly performing post‐translational modifications on downstream substrates at the protein level. Thus, TOR forms a regulatory mode that integrates nutrient, growth factor, and energy signals to orchestrate cell growth and metabolism (Chantranupong et al., 2015; Cornu et al., 2013; Dobrenel et al., 2013; Laplante & Sabatini, 2009; D. Li et al., 2022; Robaglia et al., 2012; Wullschleger et al., 2006; Yuan et al., 2020).

TOR in eukaryotes comprises five distinct domains. Each domain has specific functions, and alterations in these domains can affect TOR activity (Baretić & Williams, 2014). First is the huntingtin, EF3, PP2A A subunit, and TOR1 (HEAT) repeat domain, named after its initial discovery in huntingtin, EF3, the A subunit of PP2A, and TOR1 proteins. It mainly mediates protein‐protein interactions and serves as a core component of the N‐terminal domain of the TOR protein (Sauer et al., 2013; Yang et al., 2013). The FAT domain (FRAP‐ATM‐TRRAP domain) is a conserved domain present in PI3K‐related kinase family proteins (including TOR), located upstream of the kinase catalytic domain. This domain not only maintains the overall conformational stability of the TOR protein but also mediates the binding of TOR to regulatory factors and substrates, playing a crucial role in the regulation of kinase activity (Anderson et al., 2005). The FRB domain (FKBP12‐rapamycin‐binding domain) is a unique functional domain of the TOR protein and acts as the binding site for the rapamycin‐FKBP12 complex. When rapamycin forms a complex with FKBP12 and binds to the FRB domain, it inhibits the kinase activity of TOR. Therefore, this domain is a key target for studying the function of the TOR signaling pathway (Helliwell et al., 1994). The spatial arrangement of the FAT and FRAP‐ATM‐TRRAP C‐terminal domain domains, namely, the FRAP, ATM, and TRRAP domains and FRAP, ATM, and TRRAP C‐terminal domains, respectively, exposes the kinase domain, which supports its catalytic function. The kinase domain possesses serine/threonine kinase activity and regulates downstream signaling by phosphorylating substrates (Anderson et al., 2005; Bartel & Fink, 1994). In the current reports, the downstream targets of TOR include ribosomal protein S6 kinase (S6K), ribosome protein small subunit 6 (RPS6), type 2A‐phosphatase‐associated protein 46 Kd (α4), E2Fa transcription factor (E2F3), repressor of LRR‐extensin 1 (ATPBD3), and translationally controlled tumor protein (TCTP). mTORC1 mainly controls gene translation through its substrates S6K and eukaryotic initiation factor 4E binding protein 1 (4E‐BP1) (Reinke et al., 2004). RAP‐insensitive mTORC2, composed of mTOR, mLST8, and the rapamycin‐insensitive companion of mTOR (RICTOR), exerts its function through the phosphorylation of PKB/Akt (protein kinase B, PKB/Akt) (Cornu et al., 2013; Laplante & Sabatini, 2012; Wullschleger et al., 2006).

Nitrogen (N) is an indispensable nutrient for plant growth. Under low‐nitrogen stress, plants undergo various physiological and biochemical responses, and adaptive changes occur in various organs (Tulin et al., 2021). The growth patterns of roots and shoots in plants under low‐nitrogen stress differ from those under N‐sufficient conditions. N is reabsorbed and redistributed between shoots and roots under low‐nitrogen stress, which leads to rapid root growth, and the growth of shoots and true leaves is significantly inhibited (Moreno et al., 2020; Ruffel et al., 2011). A growing body of evidence indicates that TOR is involved in plant responses to low‐nitrogen stress (L. Song et al., 2022). Autophagy, as an important pathway for plants to cope with nitrogen deficiency (Díaz‐Troya et al., 2008), is a downstream target of TOR. TOR can regulate the initiation of autophagy by affecting the expression of ATG genes or directly phosphorylating ATG proteins (D. Li et al., 2022; Mugume et al., 2020; Yao et al., 2023). However, the composition and regulatory role of the TOR signaling pathway in perennial woody plants (e.g., apple) under low‐nitrogen stress remain unclear.

Apple is a widely cultivated fruit tree with significant nutritional and economic value. The Loess Plateau, located in the arid and semi‐arid regions of Northwest China, is one of the major apple‐producing areas. However, apple plants in this region are exposed to various abiotic stresses, including N availability. TOR plays essential roles in N signal perception and autophagy modulation, and it is evolutionarily conserved across eukaryotes (Menand et al., 2002; Montané & Menand, 2013; Xiong et al., 2013). The lack of systematic research on this topic had driven us to identify components of the TOR pathway in apple. We performed homologous alignment, analyzed their expression patterns under low‐nitrogen stress, and verified the MdTOR‐ATG interactions. This study aims to identify the conserved members of the TOR signaling pathway in apple and provide a theoretical basis for exploring their responses to low‐nitrogen stress.

Core Ideas

  • A total of 14 members in the target of rapamycin (TOR) pathway were identified in the apple genome.

  • TOR signal members respond to low nitrogen stress.

  • MdTOR interacts with multiple MdATGs in addition to conserved MdATG13.

2. MATERIALS AND METHODS

2.1. Genome‐wide identification and physicochemical analysis of TOR signaling pathway members

Based on previously reported gene IDs of TOR signaling pathway members in Arabidopsis, the amino acid sequences of TOR signaling pathway member proteins were retrieved and downloaded from the Arabidopsis database (http://www.arabidopsis.org/). Subsequently, these sequences were utilized as query sequences for comparison with the corresponding protein sequences retrieved from the Golden Delicious Genome Database in the GDR (genome database for rosaceae) database using the BLASTP tool (https://www.rosaceae.org/). The Expasy (expert protein analysis system) website (https://web.expasy.org/protparam/) was then utilized for the prediction and analysis of the physicochemical properties of TOR signaling pathway member proteins.

2.2. Multiple sequence alignment analysis

Based on the amino acid sequences of Arabidopsis TOR pathway members, a BLASTP homology search was performed on the National Center for Biotechnology Information (NCBI) website (https://www.ncbi.nlm.nih.gov/), and the homologous sequences of the proteins from different species were downloaded. The homologous proteins of TOR signaling members from five species, Homo sapiens, Arabidopsis thaliana, Malus domestica, Saccharomyces cerevisiae, and Gossyp hirsutum, were then subjected to multiple sequence alignment using DNAMAN9 software.

2.3. Calculation of Ka/Ks ratios and phylogenetic tree construction

Homologous sequences of the target genes were retrieved from the Phytozome database (https://phytozome‐next.jgi.doe.gov/) and NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/). A total of 17 representative species were selected, including humans, yeast, Arabidopsis thaliana, and Oryza sativa, as well as core species from the families Solanaceae, Fabaceae, and Rosaceae. These species cover key taxa such as animals, dicotyledons, and monocotyledons so as to ensure comprehensive evolutionary coverage.

Based on the aligned coding sequence (CDS) sequences of the target genes, a phylogenetic tree was constructed using the maximum likelihood (ML) method in MEGA 11 software. The best‐fit evolutionary model (GTR+G+I) was selected using the ModelFinder tool integrated in MEGA 11, based on the Akaike Information Criterion. The ML tree was generated with 1000 bootstrap replicates to assess the reliability of each node.

To evaluate the evolutionary pressure acting on the target genes, the non‐synonymous substitution rate (Ka) and synonymous substitution rate (Ks) were calculated in this study. The specific procedures were as follows: first, the CDS and protein sequences of the target genes from different species were aligned using ClustalX 2.1 software with default parameters, followed by manual correction to ensure the accuracy of the alignment results. Then, the corrected CDS and protein sequences were imported into TBtools software and analyzed using the Simple non‐synonymous substitution rate/synonymous substitution rate (Ka/Ks) Calculator tool embedded in it. The YN00 model was used to estimate Ka and Ks values, and the Ka/Ks ratio was used to infer the selection pressure acting on the genes (Ka/Ks > 1: positive selection; Ka/Ks = 1: neutral selection; Ka/Ks < 1: purifying selection).

2.4. Gene and protein structure analysis

The gene structure annotation general feature format version 3 (GFF3) file for Golden Delicious apple was obtained from the GDR database, and the gene structure annotation GFF3 file for Arabidopsis was acquired from http://plants.ensembl.org/index.html. Subsequently, the MEME (multiple Em for Motif Elicitation) website (https://meme‐suite.org/meme/index.html) and the CD‐batch tool in NCBI was used to analyze the conserved protein domains of TOR signaling pathway member proteins. Additionally, TBtools software was utilized to visualize the exons, introns, and conserved motifs of apple TOR signaling pathway member genes by integrating gene annotation data, MEME, and conserved domain predictions using the Gene structure viewer function (C. Chen et al., 2020). The Advanced Circos and Graphics function in TBtools software were used to perform collinearity and chromosome location analysis. Furthermore, the SWISS‐MODEL Workspace tool in the Expasy database (https://swissmodel.expasy.org/interactive) was utilized for the online prediction of the protein tertiary structures of apple and Arabidopsis TOR signaling pathway members.

2.5. Plant material and treatments

In this experiment, Malus hupehensis seedlings were used. Following surface sterilization, the Malus hupehensis seeds were subjected to 4°C and stored for approximately 2 months. Subsequently, the seeds were sown in nutrient bowls containing a mixed culture medium (soil: vermiculite: perlite, V/V/V = 3:1:1), and they were consistently managed according to Gong et al. (2017). When the seedlings had 5–6 true leaves, those with uniform growth were then selected and fixed to a foam board using a sponge. The seedlings were then placed in 5 L 1/2 Hoagland hydroponic potted plants with an N concentration of 0.2 mM for the low‐nitrogen treatment. The pH of the nutrient solution was adjusted to 6.0 ± 0.1 using phosphoric acid (the nitrogen concentration in normal hydroponic culture is 6 mM). The dissolved oxygen concentration of the solution was maintained at 8.0–8.5 mg L−1 using a dissolved oxygen controller, and the nutrient solution was replaced every 5 days. The hydroponic potted plants were housed in a hydroponic chamber with a diurnal temperature of 23°C–25°C and a nocturnal temperature of 15°C–18°C. Fluorescent lamps served as the light source and provided light for 14 h per day at a light intensity of 160 µmol·m−2·s−1. Root samples were collected at 0, 6, and 12 h, and 1, 2, 3, 4, 5, 6, and 7 days under low‐nitrogen treatment. These samples were promptly frozen in liquid N and preserved at −80°C for subsequent analysis.

2.6. Expression analysis of genes in the apple TOR pathway in response to low‐nitrogen stress

The quantitative polymerase chain reaction (qPCR) analysis was used to assess the transcription levels of various genes in the TOR signaling pathway under low‐nitrogen treatment. The root tissue samples were thoroughly ground in liquid N, and RNA was extracted using the Wolact Plant Genomic DNA Purification Kit (Wolact). The first‐strand cDNA was synthesized using the Prime Script TM RT Reagent Kit with gDNA Eraser (Perfect Real Time) (Takara). The analysis was performed using the Light Cycler 96 quantitative instrument (Roche) with a SYBR Premix Ex Taq TM II kit (Takara) as the dye, and malate dehydrogenase was used as the reference gene (Gong et al., 2017). The relative expression levels of the genes were calculated using the 2−∆∆CT method, and reactions for each sample were run in triplicate. The gene primer sequences are provided in Table S1.

2.7. Yeast two‐hybrid (Y2H) analysis

CDSs of MdTOR1, MdTOR2, and the ATG genes ATG2, ATG4, ATG5, ATG8c, ATG8i, ATG10, ATG13a, and ATG13b were cloned from the cDNA of “Golden Delicious.” Because these sequences were long, MdTOR1 and MdTOR2 were cloned into five segments according to their domains (Figure S1). The primer sequences used for gene amplification are shown in Table S1. After confirmation by sequencing, 10 fragments of MTOR1 and MdTOR2 were inserted into PGBKT7 to construct the recombinant BK vector. The autophagy‐related genes were inserted into PGADT7 to construct the recombinant AD vector (Table S1). The MdTOR1 and MdTOR2 recombinant BK vectors were then co‐transformed into the Y2H receptor with the recombinant AD vectors of eight autophagy‐related genes, respectively, according to the yeast transformation kit instructions; the transformed receptive state was cultured on different nutrient‐deficient SD media. After 3 days, the resistant colonies were diluted 10, 100, and 1000 times with sterile water and recultured on SD‐Leu/‐Trp medium. After 3 days, the resistant colonies were diluted with sterile water and cultured on SD‐Leu/‐Trp/‐his/‐ade + x‐α‐gal medium (5‐Bromo‐4‐chloro‐3‐indolyl‐α‐D‐galactopyranoside).

3. RESULTS

3.1. Identification of the key components of the apple TOR signaling pathway

Given the high conservation of the TOR signaling pathway across humans, yeast, and Arabidopsis as established in prior studies (Y. Song et al., 2017), key components of the TOR signaling pathway in apple were identified through a comparative analysis of sequences from Arabidopsis and humans with the apple genome database. The gene sequences encoding the core components of the TORC1 complex include TOR, RAPTOR, and LST8. Specific proteins of the TORC2 complex, such as RICTOR and SIN1, were not detected in the apple genome. The downstream targets of TOR in the apple genome include several core members with distinct functions (Table 1). Specifically, S6K and α4 participate in protein translation, RPS6 regulates ribosome biogenesis, and additional targets include E2F3, ATPBD3, and TCTP. Notably, SGK, TSC1, TSC2, VPS34, and 4EBP1 were not identified as TOR downstream targets in apple.

TABLE 1.

The putative components of target of rapamycin (TOR) signaling pathway in apple genome.

Proteinname Mammals Yeast Arabidopsis Apple
SIN1 √ √
RICTOR √ √
PI3Kα √ √
PI3Kβ √ √
PI3Kγ √ √
TSC1 √
TSC2 √
IRS √
SGK1 √ √
Akt √ √
4EBP1 √ √ √
AMPK √ √ √ √
LKB1 √ √ √ √
mLST8 √ √ √ √
RAPTOR √ √ √ √
mTOR √ √ √ √
FKBP12 √ √ √ √
PDK1 √ √ √ √
S6K √ √ √ √
RPS6 √ √ √ √
α4 √ √ √ √
E2F3 √ √ √ √
ATPBD3 √ √ √ √
TCTP √ √ √ √
PTEN √ √ √ √
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We further analyzed the sequences encoding these 14 members, and two sequences encoding TOR homologous proteins were identified in the apple genome TOR signaling pathway, which were denoted as MdTOR1 (MD00G1031100) and MdTOR2 (MD05G1107800). Two sequences encoding RAPTOR homologous proteins were identified, and these were denoted as RAPTOR1 (MD04G1169600) and RAPTOR2 (MD12G1183600). Two sequences encoding LST8 homologous proteins were identified as LST8‐1 (MD12G1066500) and LST8‐2 (MD14G1066600). Only one sequence encoding the FKBP12 (MD04G104740) protein was found in the apple genome. In the upstream target genes of TOR, two sequences encoding PDK1 homologous proteins were found, and these were denoted as PDK1‐1 (MD07G1165300) and PDK1‐2 (MD01G1098600). Two sequences encoding LKB homologous proteins were identified as CIPK12‐1 (MD05G1298800) and CIPK12‐2 (MD10G1278300). Two genes encoding AMP homologous proteins were also found, and these were designated as KIN1 (MD17G1053700) and KIN2 (MD09G1056200). In the downstream targets of TOR, three sequences encoding S6K homologous proteins were identified, and these were denoted as S6K1 (MD04G1176100), S6K2 (MD06G1183400), and S6K3 (MD12G1190400). Two sequences encoding α4 homologous proteins were found and denoted as TAP46‐1 (MD01G1217800) and TAP46‐2 (MD07G1287100). Two sequences encoding RPS6 homologous proteins were identified as RPS6A1 (MD15G1372800) and RPS6A2 (MD08G1186900). Two sequences encoding E2F3 homologous proteins were also found and designated as E2FA1 (MD12G1037100) and E2FA2 (MD14G1036100). Two sequences encoding ATPBD3 homologous proteins were identified as ROL1 (MD08G1004800) and ROL2 (MD15G1004300). Two sequences encoding TCTP homologous proteins were identified as TCTP1 (MD13G1167500) and TCTP2 (MD13G1167600). Two sequences encoding PTEN homologous proteins were identified as PTEN1 (MD09G1060900) and PTEN2 (MD17G1056300) (Table S2). In sum, 14 members and 28 different genes were found to be involved in the TOR signal pathway in the apple genome.

3.2. Characteristics of members of the TOR pathway in the apple genome

The ExPASy online analysis tool was used to predict the length, molecular weight, and isoelectric point of the amino acid sequences of the proteins encoded by TOR signaling pathway members in the apple genome (Table 2). The largest proteins in the TOR pathway were TOR1 and TOR2, and they comprised 2437 aa and 2465 aa, respectively; their proteins were over 270 kDa. The other component of the TOR complex is RAPTOR. RAPTOR1 and RAPTOR2 in the apple genome comprised 1337 and 1348 aa, respectively, and their proteins were all approximately 147 kDa. The other proteins ranged from 112 aa (FKBP12) to 515 aa (KIN1), and their molecular weights ranged from 12.03 to 58.95 kDa. The isoelectric points of each member ranged from 4.55 (TCTP1 and −2) to 10.7 (RPS6A1 and −2), indicating that proteins involved in the apple TOR pathway included acidic, neutral, and basic proteins, but most were acidic. Most proteins were localized to the cell nucleus. TORs were localized to the chloroplast, FKBP12 was localized to the vacuole, and TCTPs were localized to the cytoplasm. Most members were derived from whole‐genome or segmental duplications, and only a few were derived from dispersed and tandem duplications. TOR1, S6K2, and FKBP12 were derived from dispersed duplications; TCTP2 was derived from tandem duplications; and others were derived from whole genome duplication (WGD) or segmental duplications.

TABLE 2.

Gene features of members in target of rapamycin (TOR) signaling pathway in apple genome.

Name GDR No. Chromosome No. CDS (bp) Amino acid MW (kDa) Ip Subcellular localization Duplications
MdTOR1 MD00G1031100 Chr00:5158945.8185116 7385 2437 273.81 6.74 Chloroplast Dispersed
MdTOR2 MD05G1107800 Chr05:43227443.43229267 7416 2465 276.88 6.19 Chloroplast WGD or segmental
MdCIPK12‐1 MD05G1298800 Chr05:43227443.43229267 1497 498 55.56 8.21 Nucleus WGD or segmental
MdCIPK12‐2 MD10G1278300 Chr10:36891712.36894607 1491 496 55.31 7.58 Nucleus WGD or segmental
MdKIN1 MD17G1053700 Chr17:4171189.4175804 1548 515 58.95 8.4 Nucleus WGD or segmental
MdKIN2 MD09G1056200 Chr09:3719820.3723909 1371 456 52.46 6.52 Nucleus WGD or segmental
MdLST8‐1 MD12G1066500 Chr12:7848064.7851740 957 318 36.50 5.88 Nucleus WGD or segmental
MdLST8‐2 MD14G1066600 Chr14:7256884.7262534 831 276 31.10 5.97 Nucleus WGD or segmental
MdRPS6A1 MD15G1372800 Chr15:45530543.45532750 750 249 28.36 10.7 Cytoplasmic/nuclear WGD or segmental
MdRPS6A2 MD08G1186900 Chr08:23541678.23543864 750 249 28.32 10.7 Cytoplasmic/nuclear WGD or segmental
MdRAPTOR1 MD04G1169600 Chr04:26023162.26033921 4014 1337 147.05 5.97 Nucleus WGD or segmental
MdRAPTOR2 MD12G1183600 Chr12:26367345.26378184 4047 1348 147.86 5.92 Nucleus WGD or segmental
Mds6k1 MD04G1176100 Chr04:26701571.26705896 1434 478 53.71 6.2 Nucleus WGD or segmental
Mds6k2 MD06G1183400 Chr06:32180859.32178043 1410 469 52.00 6.98 Nucleus Dispersed
Mds6k3 MD12G1190400 Chr12:27318629.27322875 1440 480 53.92 6.12 Nucleus WGD or segmental
MdE2FA1 MD12G1037100 Chr12:3994051.3999058 1410 469 51.81 5.25 Nucleus WGD or segmental
MdE2FA2 MD14G1036100 Chr14:3249510.3253868 1413 470 51.80 5.37 Nucleus WGD or segmental
MdFKBP12 MD04G1047400 Chr04:5534572.5537445 339 112 12.03 6.71 Vacuole Dispersed
MdPDK1‐1 MD07G1165300 Chr07:24018720.24023252 1467 488 54.45 8.06 Nucleus WGD or segmental
MdPDK1‐2 MD01G1098600 Chr01:21224747.21229397 1467 488 54.69 8.92 Nucleus WGD or segmental
MdPTEN1 MD09G1060900 Chr09:4157990.4160716 1242 413 47.84 8.43 Nucleus WGD or segmental
MdPTEN2 MD17G1056300 Chr17:4445206.4448133 1242 413 47.93 7.87 Nucleus WGD or segmental
MdROL1 MD08G1004800 Chr08:442149.445054 1053 357 40.47 8.9 Chloroplast/nucleus WGD or segmental
MdROL2 MD15G1004300 Chr15:257717.260249 966 357 40.37 8.72 Chloroplast/nucleus WGD or segmental
MdTAP46‐1 MD01G1217800 Chr01:31034620.31037491 1221 406 45.98 5.03 Nucleus WGD or segmental
MdTAP46‐2 MD07G1287100 Chr07:34875138.34878040 1221 406 45.93 5.06 Nucleus WGD or segmental
MdTCTP1 MD13G1167500 Chr13:13558011.13560260 507 168 18.87 4.55 Cytoplasm WGD or segmental
MdTCTP2 MD13G1167600 Chr13:13560167.13571249 507 168 18.87 4.55 Cytoplasm Tandem
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Abbreviations: GDR, genome database for rosaceae; WGD, whole genome duplication.

3.3. Structure analysis of TOR pathway members in the apple genome

To investigate the evolutionary relationships and functional conservation of apple TOR proteins across different species, we selected homologous proteins from species including Malus domestica, Homo sapiens, Arabidopsis thaliana, Saccharomyces cerevisiae, and Gossypium hirsutum for homology alignment of their kinase domains. Additionally, phylogenetic tree construction and evolutionary selection pressure analysis (Ka/Ks) were performed on TOR across multiple species. The results showed that the TOR proteins in plant species shared highly similar conserved kinase domains, and the similarity rate was approximately 96.81%. Their similarity with homologous sequences in humans and yeast was approximately 93.29% and 89.24%, respectively (Figure 1A), indicating that TOR has a highly conserved kinase domain. The phylogenetic tree constructed based on the ML method demonstrated that all plant TOR proteins were clustered into two major clades, namely, monocotyledons and dicotyledons. MdTOR1 and MdTOR2 were grouped into the same clade with TOR from Rosaceae species, including Crataegus pinnatifida, Prunus persica, and Fragaria vesca (bootstrap value = 100) (Figure 1B). This suggests that TOR genes across different species have close evolutionary relationships, and TOR may have undergone functional specialization during the evolution of Rosaceae species. Results of evolutionary selection pressure analysis revealed that the Ka/Ks ratios of all plant gene pairs were <1 (0.07–0.25), indicating that MdTOR has experienced strong purifying selection during evolution and possess highly conserved functions (Table S3). In summary, these findings are consistent with the biological role of TOR as a core signaling pathway kinase. The same pattern was also observed for other proteins among these species. The lowest protein sequence similarity among the five species (26.04%) was observed for CIPK12 (Figure S2), and the highest protein sequence similarity (81.06%) was observed for RPS6 (Figure S3). For the other 11 members, the protein sequence similarity among the five species was 61.44% (RAPTOR, Figure S4), 73.23% (TCTP, Figure S5), 72.57% (LST8, Figure S6), 70.51% (FKBP12, Figure S7), 69.78% (ROL, Figure S8), 53.64% (PTEN, Figure S9), 63.31% (TAP46, Figure S10), 64.72% (PDK, Figure S11), 43.76% (E2FA, Figure S12), 59.31% (KIN, Figure S13), and 56.76% (S6K, Figure S14).

FIGURE 1.

FIGURE 1

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Homology analysis of apple target of rapamycin (TOR) proteins. (A) Amino acid sequence alignment of the TOR kinase domain from Malus domestica with those from other representative organisms: Homo sapiens (human); Arabidopsis thaliana (Arabidopsis); Malus domestica (apple); Saccharomyces cerevisiae (yeast); Gossypium hirsutum (upland cotton). (B) Phylogenetic tree of TOR proteins among different species, with Rosaceae plants highlighted in red.

We further analyzed the gene structures and protein motifs of all the members, and these were visualized using TBtools. MdTOR2 was the longest, and MdTCTP2 was the shortest. CIPK12s had only one exon, and the others had at least 5 exons. The TORs had the most exons, and there were 55 exons in its genomic sequence (Figure 2).

FIGURE 2.

FIGURE 2

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Gene structure and conserved motifs analysis diagram of target of rapamycin (TOR) signaling pathway genes in apple. CDS, coding sequence; UTR, untranslated region.

Although the members in the TOR pathway belonged to different protein families, some of them also shared the same protein motif. The conservation scores of these motifs are all >0.6, indicating that they are motifs with relatively high conservation. For example, 18 out of the total 27 proteins had motif 9. CIPKs, KINs, S6Ks, and the 2 PDKs had the most similar protein motifs; they all had motifs 4, −2, −1, −5, −3, and −6. The differences were that CIPKs and PDKs also had motif 9, and KINs had motif 8. TORs had motifs 7 and 9; RAPTORs and TCTPs only had motif 7 (Figure 3).

FIGURE 3.

FIGURE 3

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Schematic diagram of 10 conserved motifs contained in apple target of rapamycin (TOR) signaling pathway proteins. IC, information content; CS, conservation score.

To clarify the structures of proteins involved in the TOR signaling pathway based on the apple genome, we analyzed the tertiary structures and conserved domains of all the members and generated the corresponding structure diagrams. The results showed that all the proteins contained α‐helices, β‐sheets, and random coils (Figure 4A). In addition, the kinase domain had different active sites in most of the members, suggesting that the signaling cascade involved in the TOR pathway might be a phosphorylation cascade (Figure 4B).

FIGURE 4.

FIGURE 4

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Analyze of target of rapamycin (TOR) signaling pathway genes in apple proteins. (A) Diagram of the tertiary structure. (B) The protein domain predicted, with red is the active site. FAT, FRAP‐ATM‐TRRAP domain; FATC, FRAP‐ATM‐TRRAP C‐terminal domain.

3.4. Chromosome distribution and collinearity analysis of members in the TOR pathway in the apple genome

With the exception of TOR1, which could not anchor to any of the 17 apple chromosomes, the other 27 sequences were distributed on 13 different chromosomes. Chr 12 contained the highest number of genes (4 genes); chr 4 contained 3 genes. Chr 1, −5, −7, −8, −9, −13, −14, −15, and −17 each contained two genes. Ch 6 and −10 had the fewest genes, with only 1 gene each. No members were found on chr 2, −3, −11, and −16 (Figure 5).

FIGURE 5.

FIGURE 5

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Chromosome location of target of rapamycin (TOR) signaling pathway genes in apple.

Collinearity analysis was performed on TOR signaling pathway members in apple; a heatmap was used in the first circle to depict the chromosome density, and a line plot was used in the second circle to represent the chromosome density. Duplicated regions between apple TOR signaling pathway members and other genes were denoted by different colored lines connecting the genes. Segmental duplication of MdTOR2 was observed on chromosome 10; segmental duplications were also observed for MdS6K1 and MdS6K3. Furthermore, segmental duplication of MdTCTP1 was observed on chromosome 16, and segmental duplications of MdRPS6A1 and MdRPS6A2 were observed on chromosome 15. All other genes demonstrated evidence of segmental duplication, with the exception of MdFKBP12, MdTCTP2, MdTOR1, and MdS6K2 (Figure 6A).

FIGURE 6.

FIGURE 6

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Collinearity analysis of target of rapamycin (TOR) signaling pathway genes. (A) Synteny analysis of TOR signaling pathway genes in apple. (B) Synteny analysis of TOR signaling pathway genes between apple and Arabidopsis.

Comparative genomics was employed to investigate the evolutionary relationships between Arabidopsis and apple (Figure 6B). The results showed that 10 out of the 28 members in the TOR pathway in apple had 18 pairs of collinear relationships with Arabidopsis (Table S4). The majority of them showed only a single pair of collinear relationships with Arabidopsis, and MdPDK1‐1 had two pairs of collinear relationships between apple and Arabidopsis; MdKIN1 and MdKIN2 each had three pairs of collinear relationships, and MdCIPK12‐1 had four pairs of collinear relationships with Arabidopsis. This suggested that members in the TOR signaling pathway from different species may have originated from the same ancestor and play similar roles in plants.

3.5. The expression patterns of members in the TOR pathway in apple under low‐nitrogen stress

The expression patterns of members in the TOR signaling pathway were examined in the roots of Malus hupehensis subjected to low‐nitrogen treatment. We collected samples at 0, 6, and 12 h, and 1, 2, 3, 4, 5, 6, and 7 days and performed qPCR analysis. The results were visualized in the form of a heatmap using TBtools. As shown in Figure 7, most of the detected genes were first gradually induced to a peak by low‐nitrogen stress, and then their expression level decreased; their highest levels were reached at 3 or 4 days. LST8‐1, KIN2, RPS6A1, RAPTOR2, TOR1, PDK1‐2, E2FA2, PTEN1, and S6K3 reached their highest expression levels at 3 days and were 1.7, 1.7, 1.4, 2.0, 1.6, 1.2, 1.7, 2.0, 1.7, 1.9, and 2.2 times their initial levels, respectively. At 4 days, the expression levels of S6k2, TAP46‐1, TAP46‐2, CIPK12‐1, CIPK12‐2, KIN1, and RPS6a2 were 2.9, 2.7, 2.1, 3.3, 2.5, 3.7, and 1.4 times their initial levels, respectively. These results indicated that the TOR pathway was also probably involved in the response to low nitrogen in apple plants.

FIGURE 7.

FIGURE 7

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Relative expression patterns of apple target of rapamycin (TOR) signaling pathway genes in response to low N stress.

3.6. TORs in the apple genome interact with several autophagy‐related proteins (ATGs)

TOR is the conserved upstream regulator of autophagy by phosphorylating ATG13. We also found that TOR might directly interact with ATGs in Malus hupehensis during the response to low‐nitrogen stress (D. Li et al., 2022). To confirm the interaction between TOR and ATGs, we employed several ATGs in the apple genome and conducted Y2H analysis. The CDSs of both TOR1 and TOR2 were nearly 7500 bp; given the difficulty of cloning the whole CDS of TORs into the target vector, we cut them into five different segments according to their domains. As shown in Figure 8, eight ATGs, including ATG2, ATG4a, ATG5b, ATG8c, ATG8i, ATG10, ATG13a, and ATG13b, were found to directly interact with different TOR truncations in the Y2H assay; the others could not interact with any of the TOR truncations. Although the similarity in the sequences and domains of TOR1 and TOR2 was high, they did not share the same downstream ATGs. They even interacted with the same ATG, and the functional domains were different. For example, TOR1, but not TOR2, can interact with ATG2. The third and the fifth truncations of TOR2 and the first, third, and fourth truncations of TOR1 interacted with ATG4a. ATG8c can interact with each truncation of both TOR1 and TOR2. In addition, the third truncation of TOR1 can interact with all the detected ATGs. These results suggested that except for ATG13, TOR can directly interact with ATGs through its various domains. There might be other mechanisms underlying the regulatory relationships between TOR and autophagy in apple plants.

FIGURE 8.

FIGURE 8

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MdTOR1 and MdOR2 directed interact with proteins involved in Autophagy pathway.

4. DISCUSSION

TOR, a conserved serine/threonine protein kinase in eukaryotes, plays a pivotal role in regulating cell growth and metabolism in response to environmental cues. In plants, an increasing number of studies have clarified the significance of the TOR signaling pathway in controlling plant growth and stress responses across various developmental stages (Liu & Xiong, 2022). In nutrient metabolism, TOR responds to sugar signals and senses changes in cellular metabolism and bioenergy status to regulate plant energy signal transduction. Once activated by glucose, TOR phosphorylates and activates the transcription factors E2Fa/E2Fb. This activation enhances cell division in the root meristem and shoot apical meristem, thereby promoting root growth and true leaf formation, respectively (L. Li et al., 2015).

TOR also mediates crosstalk between sugar signaling and brassinosteroid signaling. Glucose‐activated TOR can inhibit autophagy to stabilize BZR1 (a positive regulator of brassinosteroid signaling), thereby promoting cell growth in the hypocotyl (Zhang et al., 2016). Key upstream signals activated by TOR include organic N molecules. The signaling pathway with TOR as the core includes multiple components; the members in its conserved signaling pathway have been analyzed in humans, yeast, Arabidopsis, and microalgae, and they have similar conserved members in their respective signaling pathways (Mallén‐Ponce et al., 2022; Otsubo & Yamamato, 2008; Tatebe & Shiozaki, 2017). In this study, we aligned sequences from Arabidopsis and humans with the apple genome database and identified key members of the TOR signaling pathway in apple.

The composition of TOR signaling pathways in plants significantly differed from that in humans or yeast. In the apple genome, a total of 14 members and 28 genes were identified. Despite the presence of two TOR proteins in apples, key components of the TORC2 pathway are absent, and only the TORC1 pathway was detected, which is consistent with the results of studies of Arabidopsis and cotton (Y. Song et al., 2017). Alignment of these genes with their homologs in Arabidopsis revealed that their sequences were highly similar, indicating that the TOR pathway was highly conserved in apple and Arabidopsis. In addition, 10 of the TOR signaling pathway members in apple had 18 collinear relationships with Arabidopsis based on an inter‐species collinearity analysis, indicating their possible common ancestry and similar functions in plants. There were seven genes encoding the core components of the TORC1 complex, six genes encoding the upstream components, and 15 genes encoding downstream components in the apple genome. Both TOR and RAPTOR in Arabidopsis have a single clone, and there were two sequences encoding them in the apple genome. LST8, PDK1, PTEN1, ROL, KIN, TAP46, CIPK12, TCTP, PTEN, and E2FA were also encoded by two sequences in the apple genome. S6K was encoded by three sequences. FKBP12 was identical in the Arabidopsis and apple genome and comprised a single CDS. Genes encoding members of the TOR signaling pathway were higher in apple than in Arabidopsis, and most of them were derived from WGD or segmental duplications; only TOR1, S6K2, FKBP12, and TCTP2 were derived from dispersed and tandem duplications. In the apple genome, 70% and 26.1% of the duplications stemmed from WGD and tandem duplications, respectively (Yuet et al., 2015). Previous studies have shown that several gene families such as MYB, NAC, and DREB in apple are amplified by WGD and tandem duplications (Cao et al., 2013; Su et al., 2013; Zhao et al., 2012). Gene replication and tandem repeats play an important role in the distribution of genes in plants (Cannon et al., 2004; Kong et al., 2007). TOR signaling pathway members in Arabidopsis were distributed across five chromosomes; in apple, they were unevenly distributed across 13 chromosomes. Gene duplications contribute to genomic expansion and the diversification of gene functions, and genes involved in transcriptional regulation, signal transduction, and stress responses tended to have paralogs (Maere et al., 2005; Panchy et al., 2016). For example, the aluminum‐activated malate transporters (ALMTs) are related to abiotic stress. In Arabidopsis, the AtALMT family has 14 members, most of which are associated with aluminum tolerance (Hoekenga et al., 2006), and the same was the case for MsALMT1 from Medicago sativa and HlALMT1 from Holcus lanatus (Q. Chen, Wu, et al., 2013; Z. C. Chen, Yokosho, et al., 2013). However, OsALMT1 in rice is involved in the responses to salt stress and osmotic stress (Lu et al., 2019). In addition, sucrose transporters or carriers (SUTs or SUCs) function as sucrose/H+ symporters. MdSUT2 in apple plants is a homologous gene of the Arabidopsis vacuolar sucrose transporter AtSUT2. AtSUT2 reduces the sensitivity to sucrose during seed germination and the seedling stage and enhances sucrose transport activity (Barker et al., 2000). MdSUT2 significantly improves the tolerance of apple calli to abiotic stresses, such as salt and drought (Ma et al., 2016). Although the TOR signaling pathway is highly conserved, the expansion of coding genes in the apple genome may support the diversification of TOR signaling functions in apple plants.

TOR is a key regulator of plant growth under low‐nitrogen stress. In nitrogen‐deprived seedlings, TOR activity is significantly inhibited, while exogenous supplementation of nitrate, ammonium, or amino acids can rapidly restore its kinase activity (L. Song et al., 2022). In this study, we found that the expression levels of most genes in the TOR pathway initially increased and then decreased, which indicates that the TOR signaling pathway responds to low‐nitrogen stress. Previous studies of apple plants showed that TOR can alter hormone metabolism and autophagy activity in apple seedlings in response to low‐nitrogen stress (D. Li et al., 2022). The inhibition of TOR activity increases plant autophagy activity; during N starvation, autophagy can degrade damaged organelles, activate N‐related proteins to promote N absorption and assimilation (Huo et al., 2020; Zhen et al., 2019). Autophagy is an important downstream pathway of TOR. TOR is a key negative regulator of the initiation of autophagy; inhibition of TOR can increase the autophagic activity of plants to adapt to nutritional stress. The gradual assembly of ATG proteins is a key step in autophagosome biogenesis during the initiation of autophagy; TOR inhibits the activation of autophagy by directly phosphorylating the Ser258 of ATG13 to regulate the formation of the ATG1‐ATG13‐ATG17 complex (Yao et al., 2023). In addition, TOR can modulate the phosphorylation of ATG14l to regulate the early process of autophagy (Matsunaga et al., 2010). An increasing number of studies have shown that TOR can regulate the process of autophagy by directly interacting with ATGs. We also found that TOR might interact with a large number of ATG proteins after constructing the protein–protein interaction network (D. Li et al., 2022). In this work, we further verified the interaction between TOR and ATGs via Y2H assays. The results showed that there were physical interactions between MdTORs and autophagy proteins such as MdATG2, MdATG4a, MdATG5b, MdATG8c, MdATG8i, MdATG10, MdATG13a, and MdATG13b. MdATG4a and MdATG5b directly interacted with the kinase domain of MdTOR2, and MdATG8c interacts with both the kinase domains of MdTOR1 and MdTOR2, indicating that they may be the phosphorylation substrates of TOR in apple plants. In addition, most of the ATGs interacted with the first, third, or fourth segments of TORs. These domains consist of a multi‐helical fold, featuring two curved layers of α‐helices arranged in a regular right‐handed superhelix. The superhelical structure provides a large solvent‐accessible surface, making it highly suitable for binding large substrates such as proteins and nucleic acids (Groves & Barford, 1999). Research indicates that RAPTOR interacts with the N‐terminal region (amino acids 1–1482) of mTOR, which contains all the HEAT motifs, and the overall structure of the N‐terminal portion of mTOR is required for its binding to RAPTOR (Kim et al., 2002). In addition, the interactions mediated by the HEAT domains are diverse. They can bind to both proteins and RNAs. eIF4G has three HEAT domains and exists in two isoforms, which may show certain selectivity biases toward different mRNAs (Jackson et al., 2010). The HEAT1 domain of DAP5 can also bind to RNAs (Marash & Kimchi, 2005). These results suggest that, aside from the conserved ATG13, TOR may regulate the activity of autophagy in multiple ways. However, which one plays the major regulatory role, TOR1 or TOR2, and how they work in regulating autophagy or other biological processes, requires further study.

It should be noted that this study focused on the identification, expression pattern analysis, and verification of interaction with ATG proteins of TOR signaling pathway members in apple. It is preliminarily speculated that the conserved members of the TOR pathway in apple may play important roles in response to low‐nitrogen stress, and TOR can also regulate ATGs. However, we have not yet conducted in‐depth investigations into the physiological functions and molecular mechanisms of these members in the TOR signaling pathway during low‐nitrogen stress responses, nor have we further verified the mechanism of TOR‐mediated regulation of ATGs. Naturally, these aspects have been incorporated into our future research agenda. Moving forward, it will be necessary to employ transgenic technology and other approaches to analyze the specific functions of these members one by one, and to verify whether MdTOR can directly phosphorylate the interacting ATG proteins through in vitro kinase activity assays, thereby constructing a comprehensive TOR regulatory network.

5. CONCLUSION

In conclusion, we identified 14 members and 28 genes involved in the TOR signaling pathway in the apple genome. They were mapped to 13 chromosomes; their protein sequence lengths ranged from 112 aa to 2465 aa, and their isoelectric points ranged from 4.55 to 10.7. Most TOR signaling pathway members are located in the cell nucleus, with a few located in the cell membrane or extracellular space. Many of them exhibit collinearity with Arabidopsis members. In addition, the majority of the members in the TOR signaling pathway in the apple genome can be induced by low‐nitrogen stress. Aside from the conserved downstream target ATG13, MdTORs can physically interact with autophagy proteins, such as MdATG2, MdATG4a, MdATG5b, MdATG8c MdATG8i, and MdATG10. This systematic analysis of the key components of the TOR signaling pathway in the apple genome reveals the diversification of the TOR signaling pathway in plants. Their expression under low‐nitrogen stress and the interaction between TOR and several ATG proteins will aid further study of the TOR pathway under low‐nitrogen stress and provide valuable reference data.

AUTHOR CONTRIBUTIONS

Dehai Liu: Writing—original draft. Siyuan Cheng: Data curation. Yanghao Long: Data curation. Qinyao Jin: Data curation. Yujie Zhou: Data curation. Fengwang Ma: Conceptualization. Xuan Liu: Writing—review and editing. Xiaoqing Gong: Conceptualization; funding acquisition.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Supporting information

Supplementary material

TPG2-19-e70215-s001.docx (859.6KB, docx)

Supplementary material

TPG2-19-e70215-s002.xlsx (19.7KB, xlsx)

ACKNOWLEDGMENTS

The authors are grateful to Xiaoqing Gong, Xuan Liu, and Fengwang Ma for their valuable input in conceptualization of the research. We are grateful to Dr. Zhihong Zhang from Shenyang Agricultural University for providing tissue‐cultured GL‐3 plants in this work. We thank Siyuan Cheng, Qinyao Jin, and Yujie Zhou for their data collection efforts. We also thank the staff members of State Key Laboratory for Crop Stress Resistance and High‐Efficiency Production/Shaanxi Key Laboratory of Apple. This study was supported by the National Natural Science Foundation of China (32472667, 32372648) and the earmarked fund for the China Agricultural Research System (CARS‐27).

Liu, D. , Cheng, S. , Long, Y. , Jin, Q. , Zhou, Y. , Ma, F. , Liu, X. , & Gong, X. (2026). Genome‐wide identification and expression analysis of TOR signaling pathway components in apple in response to low nitrogen. The Plant Genome, 19, e70215. 10.1002/tpg2.70215

Assigned to Associate Editor David Edwards.

Contributor Information

Xuan Liu, Email: gongxq0103@nwsuaf.edu.cn.

Xiaoqing Gong, Email: Lx156273@163.com.

DATA AVAILABILITY STATEMENT

The data underlying this article are available in the GDR database at: https://www.rosaceae.org/, and can be accessed with MdTOR1 (MD00G1031100), MdTOR2 (MD05G1107800), RAPTOR1 (MD04G1169600), RAPTOR2 (MD12G1183600), LST8‐1 (MD12G1066500), LST8‐2 (MD14G1066600), FKBP12 (MD04G104740), PDK1‐1 (MD07G1165300), PDK1‐2 (MD01G1098600), CIPK12‐1 (MD05G1298800), CIPK12‐2 (MD10G1278300), KIN1 (MD17G1053700), KIN2 (MD09G1056200), S6K1 (MD04G1176100), S6K2 (MD06G1183400), S6K3 (MD12G1190400) TAP46‐1 (MD01G1217800), TAP46‐2 (MD07G1287100), RPS6A1 (MD15G1372800), RPS6A2 (MD08G1186900), E2FA1 (MD12G1037100), E2FA2 (MD14G1036100), ROL1 (MD08G1004800), ROL2 (MD15G1004300), TCTP1 (MD13G1167500), TCTP2 (MD13G1167600), PTEN1 (MD09G1060900) and PTEN2 (MD17G1056300).

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Associated Data

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

Supplementary Materials

Supplementary material

TPG2-19-e70215-s001.docx (859.6KB, docx)

Supplementary material

TPG2-19-e70215-s002.xlsx (19.7KB, xlsx)

Data Availability Statement

The data underlying this article are available in the GDR database at: https://www.rosaceae.org/, and can be accessed with MdTOR1 (MD00G1031100), MdTOR2 (MD05G1107800), RAPTOR1 (MD04G1169600), RAPTOR2 (MD12G1183600), LST8‐1 (MD12G1066500), LST8‐2 (MD14G1066600), FKBP12 (MD04G104740), PDK1‐1 (MD07G1165300), PDK1‐2 (MD01G1098600), CIPK12‐1 (MD05G1298800), CIPK12‐2 (MD10G1278300), KIN1 (MD17G1053700), KIN2 (MD09G1056200), S6K1 (MD04G1176100), S6K2 (MD06G1183400), S6K3 (MD12G1190400) TAP46‐1 (MD01G1217800), TAP46‐2 (MD07G1287100), RPS6A1 (MD15G1372800), RPS6A2 (MD08G1186900), E2FA1 (MD12G1037100), E2FA2 (MD14G1036100), ROL1 (MD08G1004800), ROL2 (MD15G1004300), TCTP1 (MD13G1167500), TCTP2 (MD13G1167600), PTEN1 (MD09G1060900) and PTEN2 (MD17G1056300).

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