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Evolution of the SPX gene family and its role in the response mechanism to low phosphorus stress in self-rooted apple stock

This article has been updated



Phosphorus plays a key role in plant adaptation to adversity and plays a positive role in the yield and quality formation of apples. Genes of the SPX domain-containing family are widely involved in the regulation of phosphorus signalling networks. However, the mechanisms controlling phosphorus deficiency are not completely understood in self-rooted apple stock.


In this study, 26 members of the apple SPX gene family were identified by genome-wide analysis, and further divided into four subfamilies (SPX, SPX-MFS, SPX-EXS, and SPX-RING) based on their structural features. The chromosome distribution and gene duplications of MdSPXs were also examined. The promoter regions of MdSPXs were enriched for multiple biotic/abiotic stresses, hormone responses and typical P1BS-related elements. Analysis of the expression levels of 26 MdSPXs showed that some members were remarkably induced when subjected to low phosphate (Pi) stress, and in particular MdSPX2, MdSPX3, and MdPHO1.5 exhibited an intense response to low Pi stress. MdSPX2 and MdSPX3 showed significantly divergent expression levels in low Pi sensitive and insensitive apple species. Protein interaction networks were predicted for 26 MdSPX proteins. The interaction of MdPHR1 with MdSPX2, MdSPX3, MdSPX4, and MdSPX6 was demonstrated by yeast two-hybrid assay, suggesting that these proteins might be involved in the Pi-signaling pathway by interacting with MdPHR1.


This research improved the understanding of the apple SPX gene family and contribute to future biological studies of MdSPX genes in self-rooted apple stock.

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Phosphorus (P) is an essential macronutrient for all living organisms. Although phosphorus is abundant in the environment, it is limited due to its low dispersal rate, uptake by the soil, and conversion to organophosphorus by microorganisms. The lack of phosphorus in the soil greatly reduces the growth of plants, and has become the main constraint of agricultural production [1, 2].

Phosphorus plays a key role in plant adaptation to adversity and plays a positive role in the yield and quality formation of apples [3, 4]. According to the soil nutrient conditions in many apple producing areas, it is found that the apple orchards in China are mostly established in the mountains with barren soil layer, water and nutrient shortage and complex climate Hill field, most of the soil has a very low effective P content [5, 6]. In the process of apple cultivation, the lack of effective phosphorus in the soil will delay the growth of trees, form stiff seedlings, delay flowering and maturity, lead to falling flowers and fruits, hinder sugar transportation, and affect the yield and quality of apples. Good root morphology and physiological characteristics are the basis for the absorption and utilization of phosphorus nutrients and adaptation to soil low phosphorus environment [7]. Therefore, the selection of low phosphorus resistant apple stocks is of great significance for the sustainable development of apples.

The SPX gene family involved in many phosphate (Pi) signalling pathways and the SPX domain was first identified in a yeast gpa1 inhibitor (SYG1), a cyclin-dependent kinase inhibitor (PHO81) and human xenotropic and polytropic retrovirus receptor 1 (XPR1) [8, 9, 18]. Based on the presence of additional domains, SPX proteins can be further classified into four subfamilies: SPX proteins, SPX-EXS proteins, SPX-MFS proteins, and SPX-RING proteins [10,11,12]. The AtSPXs, CoSPXs, GmSPXs, PeSPXs, BnaSPXs, TaSPXs, OsSPXs, and ZmSPXs have been characterized [13,14,15,16,17,18,19,20]. PHOSPHATE RESPONSE 1 (PHR1) as a key regulator of transcriptional responses to Pi starvation, is a transcription factor with an MYB domain and a coiled-coil domain binding to a P1BS cis-element, which was identified in Arabidopsis thaliana [21]. The certain SPX domain-containing proteins regulated the transcriptional activity of PHR1. Under Pi deficiency, the content of cellular inositol pyrophosphate decreases, releasing PHR1 from the SPX1 and PHR1 complex to bind to the promoter of the PSI genes, thereby increasing genes transcription [22,23,24,25].

Classifying gene family members in the genome in mining biological problems related to species traits is the first step, laying a solid foundation for subsequent gene function studies and genetic transformation studies. However, the mechanisms controlling phosphorus deficiency are not clear in self-rooted apple stock. In this study, a bioinformatics analysis of SPX gene family members was performed in the apple genome, including chromosomal locations, phylogenetic relationships, gene structures, gene duplication, and cis-element analysis. We further proved that MdSPX2, MdSPX3, MdSPX4, and MdSPX6 physically interacted with MdPHR1. This study revealed the molecular function of SPX gene in apples, which can provide a theoretical basis for the breeding of low phosphorus resistance, and has great significance for the sustainable development of the apple industry.


Identification and chromosomal locations of apple SPX genes

Using 20 Arabidopsis SPX proteins as search sequences, a total of 26 apple SPX genes were identified by BlastP search in the Malus×domestica reference genome database. The accuracy of the apple SPX genes was verified based on Pfam database ( and SMART website ( [26]. The MdSPX family genes were named based on their homologs in Arabidopsis as follows: six genes belonged to the SPX subfamily, the SPX-EXS subfamily contained 12 genes, three members belonged to the SPX-MFS subfamily, and five members belonged to the SPX-RING subfamily. The gene ID, molecular weight, amino acids length and the isoelectric points (pI) of the 26 MdSPX genes were summarized in Table 1. The amino acids lengths of the 26 MdSPX proteins ranged from 262aa (MD07G1115200) to 835aa (MD00G1081000), and the predicted molecular weights varied from 30.21 kD to 96.25 kD. All proteins of these four subfamilies except MdSPX1, MdSPX2, MdSPX4, MdSPX5, MdSPX6, MdSPX-MFS1, MdSPX-MFS2, and MdNLA3 had a pI greater than seven, indicating that these proteins belonged to basic proteins (Table 1). The chromosomal distribution of the MdSPX family genes was shown in Fig. 1. Among the 26 genes in apple, 21 genes were successfully mapped onto 13 chromosomes of apple, and 5 SPX-EXS subfamily genes were mapped onto Chr0. Chromosomes 3, 4, 6, 7, 12 and 14 contained only one gene, chromosomes 2, 9, 11, 13, 15 and 17 contained two genes, while chromosome 16 contained three genes (Fig. 1).

Table 1 Description of Malus domestica SPX family genes
Fig. 1
figure 1

Distribution of MdSPX family genes on apple chromosomes

Conserved motif and gene structure analysis of SPX gene family

To preliminarily resolve the phylogenetic relationships of apple SPX proteins, we comparatively analysed the phylogenetic trees, conserved motifs and gene structures of 26 MdSPX and 20 AtSPX in detail (Fig. 2). As shown in Fig. 2A, the phylogenetic tree classified these 26 SPX proteins into four clades, which were named Clade I, Clade II, Clade III, and Clade IV corresponding to the SPX-RING subfamily, SPX-MFS subfamily, SPX subfamily, and SPX-EXS subfamily, respectively. Each clade contains MdSPX and AtSPX genes, and the proteins in the same subfamily were highly related.

Fig. 2
figure 2

Phylogenetic relationships, conserved motifs and gene structure analysis of apple and Arabidopsis SPX genes. (A) A phylogenetic tree of 26 MdSPX and 20 AtSPX protein sequences was generated using MEGA 7.0 and clustered into four branches. (B) Prediction of conserved motifs in SPX proteins was performed by MEME software. The corresponding 10 conserved sequences were referred to Figure S1. (C) Gene structure of SPXs.

For the evolutionary and functional analysis of gene family members, analysing gene structures could provide critical clues. To clarify the exon/intron structure of the SPX genes, we visualised their gene structures using GSDS (Fig. 2C). Similar gene structure patterns were presented in the same clade. Of the 26 SPX genes, members of the SPX-RING subfamily contained only six exons, whereas the number of exons ranged from nine to ten in the SPX-MFS subfamily, eight to fifteen in the SPX-EXS subfamily, and two to three in the SPX subfamily (Fig. 2C).

To further resolve the structural diversity of the SPX proteins and predict their functions, we used MEME to predict the number and composition of conserved motifs in AtSPX and MdSPX proteins (Fig. 2B). Ten different motifs were identified (Fig. S1). Similar motif distribution patterns were found among proteins belonging to the same clade. Motif 4 was present in all SPX proteins, suggesting that this motif was a characteristic motif specific to SPX family genes and might be related to the common function of SPX proteins. Some specific motifs were only present in specific clade as follows motifs 1, 3, 5, 7, 9, and 10 were only included in all members of Clade IV subfamily and not in other subfamilies, further confirming the accuracy of the subfamily division. Clade IV was the subfamily with the most motifs, containing 10 motifs, whereas the other three subfamilies contained only 1–3 motifs (Fig. 2B). Thus, the functions of these motifs need to be further investigated to understand how these proteins function.

Overall, the SPX genes that were closely related in evolutionary terms in the phylogenetic tree had similar conserved motifs and gene structures, indicating that proteins within the same subfamily may exhibit similar functions.

Phylogenetic analysis of SPX proteins in different plant species

To further explore the evolutionary relationships of SPX gene family members, we performed a CLUSTALW alignment of 157 SPX protein sequences, consisting of 26 MdSPX, 20 AtSPX, 46 TaSPX, 32 ZmSPX, 18 SlSPX, and 15 OsSPX from six different species, and constructed a phylogenetic tree using the NJ method of MEGA 7. These proteins were divided into four subfamilies (Fig. 3), of which SPX-EXS was the subfamily containing the most proteins. Monocotyledonous (Triticum aestivum, Zea mays, and Oryza. Sativa) and dicotyledonous species (Solanum lycopersicum, Malus domestica, and Arabidopsis) were clearly divided into distinct evolutionary clades. The members of the SPX-EXS and SPX-MFS subfamilies showed clear species differentiation, implying that SPX-EXS and SPX-MFS proteins might have different biological functions in distinct plants. In general, phylogenetic analysis could provide reference for the evolution and function of family genes.

Fig. 3
figure 3

Phylogenetic tree consisting of 157 SPX proteins from Arabidopsis thaliana, Solanum lycopersicum, Zea mays, Oryza sativa, Malus domestica and Triticum aestivum. SPX proteins from different species were tagged with different coloured stars

Gene duplication analysis of MdSPXs

To assess the genomic distribution and duplication of the MdSPX genes, we analysed the syntenic regions of the MdSPX genes using MCscanX software to examine the duplication of MdSPXs. As shown in Table S2, 2814 tandemly duplicated gene pairs and 11,473 segmental duplication blocks were found in the apple genome, respectively. Only one tandemly duplicated gene pair (MdPHO1.7/MD00G1081300 and MdPHO1.10/MD00G1081400) was found in the MdSPX gene family (Fig. 4; Table S2). Moreover, we also identified seven pairs of segmental duplication events of SPX genes in apple as follows MdSPX1 and MdSPX6; MdSPX4 and MdSPX5; MdPHO1.8 and MdPHO1.4; MdPHO1 and MdPHO1.1; MdPHO1.2 and MdPHO1.3; MdPHO1.8 and MdPHO1.11; MdNLA2 and MdNLA3 (Fig. 4; Table S2).

Fig. 4
figure 4

Analysis of gene duplications between chromosomes for the MdSPX genes. Different coloured lines connected segmental duplicated MdSPX gene pairs, with a total of seven segmental duplications. Small green lines marked tandemly duplicated gene pair

Cis-acting regulatory analysis of MdSPX genes

Cis-elements in promoters of genes play an essential role in the overall regulation of gene expression. There is increasing evidence that genes with similar expression patterns may share the same cis-elements. The cis-elements in the 2-kb upstream of the transcription start site of MdSPX genes were predicted using PlantCARE and PlantPAN 3.0 software (Table S3), and three main categories were classified, namely, biotic/abiotic-related stress, growth and development response-elements, and hormone response-elements (Fig. 5; Table S3). Biotic/abiotic-related stresses included light response elements (G-box, GT1-motif, I-box, Box4, GATA-motif, ARE and Sp1), low temperature response element (LTR), drought response elements (MBS, MYC, DRE core and TC-rich repeats) and damage response element (WRE3). We also found that W box elements were extensively present in several MdSPX gene promoters, suggesting that these genes might be regulated by WRKY transcription factors. Development response-elements (such as O2-site, CAT-box and RY-element) were also commonly contained in promoters. In addition, six hormone elements related to phytohormones (such as salicylic acid, methyl jasmonate and abscisic acid) were recognised. SPX genes play key roles in Pi homeostasis, and the P1BS elements (PHR1 binding site) in their promoters are essential in responding to Pi starvation. P1BS elements were notably enriched in the promoters of all MdSPX genes except MdPHO1.9 and MdPHO1.7 and multiple copies of P1BS elements were detected in these gene promoters, implying that these genes might be regulated by MdPHR1 under Pi deficiency.

Fig. 5
figure 5

Display of cis-elements in the promoters of the MdSPX genes in apple. The different colours represented the corresponding cis-element types in the MdSPX promoters and were classified into 3 main categories: biotic/abiotic-related stress, growth and development response-elements, and hormone response-elements

Expression of MdSPX genes under low pi condition

In this study, the self-rooted apple stock superior ‘12 − 2’ and ‘M9T337’ were screened for low Pi stress treatments. The results of our hydroponic experiments showed that the length of adventitious root, shoot height, and the number of adventitious root in ‘M9T337’ changed significantly under low Pi stress treatments but exhibited no significant changes in ‘12 − 2’ plants (Fig. 6A, B). The total Pi concentration, SPAD values, and anthocyanin levels in ‘12 − 2’ were higher than that in ‘M9T337’ under low-Pi conditions (Fig. 6C), indicating that ‘M9T337’ was a Pi-sensitive genotype and that ‘12 − 2’ was a Pi-tolerant genotype. The expression patterns provide essential clues for understanding the regulatory role of SPX genes. A number of MdSPX family genes were highly induced under low Pi stress condition, among which the expression levels of MdSPX2, MdSPX3, and MdPHO1.5 genes showed strong up-regulation in response to low Pi stress (Fig. 7).

Fig. 6
figure 6

Phenotypic differences between ‘12 − 2’ and ‘M9T337’ plants under phosphorus starvation. (A) Phenotypic observations of ‘12 − 2’ and ‘M9T337’ after 21 days of normal (NP: 1 mmol) and phosphorus deficiency stress (LP: 10 µmol). Scale bars = 1 cm. (B) The root length, shoot height, and the number of adventitious root between ‘12 − 2’ and ‘M9T337’ plants were measured on the 21 days after normal Pi and Pi deficiency stress treatments. (C) The total Pi concentration, SPAD values, and anthocyanin levels between ‘12 − 2’ and ‘M9T337’ plants were measured on the 21 days after normal Pi and Pi deficiency stress treatments. Samples with different letters are significantly different: P < 0.05 (Fisher’s LSD mean separation test)

Fig. 7
figure 7

Expression pattern analysis of 26 MdSPX genes in ‘12 − 2’ samples at different times of low Pi treatment (10 µmol). Total RNA was extracted from treated roots. The apple 18 S gene was used as an internal control, and each experiment included three biological replicates

Prediction of the MdSPX protein interaction network

The analysis of protein interaction network has proven to be an effective approach to study gene function. The STRING online software was used to predict the protein interaction network queried with 26 MdSPX protein sequences. MdSPX protein interaction network analysis showed that several MdSPX proteins interacted with each other, for example MdPHO1.3 might interact with MdSPX3, MdSPX4, MdSPX5 and MdSPX6 (Fig. 8). As an important transcription factor in plant phosphorus regulatory network, PHR protein plays a key role in signal transduction regulation under Pi starvation induction. PHR1 normally interacts with proteins containing the SPX domains in a Pi-dependent manner to regulate transcription. We found that phosphate starvation response (MdPHR1 and MdPHR1-like) proteins interacted with several MdSPX proteins, further highlighting the importance of SPX in maintaining phosphorus homeostasis. MdBRE1 and MdBRE1-like, the E3 ubiquitin pathway proteins, were also identified to interact with several MdSPX proteins (Fig. 8). In addition, the deoxyribodipyrimidine photo-lyase (XP_008369510.1), an enzyme that catalyzes chemical reactions, was present in the MdSPX protein interaction network. The predicted protein association network provides a meaningful reference for further studies.

Fig. 8
figure 8

Protein interaction network of MdSPXs. The network was predicted by the online software STRING. The different coloured lines represented the different types of evidence for the prediction of the interaction network

MdSPXs physically interact with MdPHR1

The protein interaction network revealed that MdPHR1 interacted with several MdSPX proteins. To verify whether MdPHR1 interacted with MdSPXs, yeast two-hybrid experiments were carried out. The recombinant plasmids AD-MdSPX1, AD-MdSPX2, AD-MdSPX3, AD-MdSPX4, AD-MdSPX5, and AD-MdSPX6 were co-transformed with BD-MdPHR1 in yeast cells, and yeast cells was observed on SD/-Trp-Leu-His-Ade medium with X-a-gal. As shown in Fig. 9, MdPHR1 was found to interact with MdSPX2, MdSPX3, MdSPX4, and MdSPX6, but not with MdSPX1, MdSPX5. To demonstrate biological significance of MdSPX and MdPHR1 interaction, we found that the expression levels of MdSPX2, MdSPX3, MdPSI1, and MdPSI2 in ‘12 − 2’ were higher than that in ‘M9T337’ in response to low Pi stress (Fig. S2), indicating that these genes might play key role in the Pi-signaling pathway in self-rooted apple stock.

Fig. 9
figure 9

Yeast-two-hybrid assays validating the interaction of MdPHR1 with the MdSPX proteins. SD, Synthetic defined; X-a-Gal, 5-bromo-4-chloro-3-indolyl-a-D-galactopyranoside


Physiological and metabolic processes in response to phosphorus deficiency in plants depend on a complex and fine-grained phosphorus signalling regulatory network in their bodies [27]. With the development of molecular biology research techniques, more and more transcription factors and phosphorus starvation-responsive genes in plant phosphorus signalling regulatory pathway have been identified, which in turn has greatly contributed to the understanding of the phosphorus signalling network. Genes of the SPX domain-containing family are widely involved in the regulation of phosphorus signalling networks. Over the past years, the role of SPX family genes in regulating the Pi signal network has been studied in Arabidopsis, wheat, rice and maize. However, functional and evolutionary research on MdSPXs has been less reported. In this study, we identified a total of 26 MdSPX genes and classified them into four subfamilies (Fig. 2; Table 1). Gene structure and motif composition can provide valuable information on the genetic relationships of multi-gene families [28, 29]. As shown in Fig. 2, SPXs shared similar gene structure and motif composition within the same group. Similar functions are usually shared by genes with similar structures and conserved motifs. MdSPX proteins were clustered into several Arabidopsis functional clades, offering useful guidance for understanding MdSPX gene function.

To explore the evolutionary relationship of MdSPX family genes with SPX genes from other species, we constructed a phylogenetic tree of 157 SPX proteins across six species and broadly grouped them into four subfamilies (Fig. 3). The apple SPX family genes were clearly classified into different evolutionary clades compared to the SPX family genes in Triticum aestivum, Zea mays, and Oryza Sativa, and the evolutionary relationships of the SPX-EXS and SPX-MFS subfamily members varied markedly among species, suggesting that these two subfamily proteins may have different roles in different plants. Gene duplication events have been shown to play an important role in the speedy expansion and evolution of gene families. Overall, 2814 tandem duplication gene pairs and 11,473 segmental duplication blocks were observed in apple genome (Table S2). In this study, only one segmental duplication events and seven tandem duplication gene pairs were identified in the MdSPX family by MCScanX (Fig. 4; Table S2).

Similar to other species, the MdSPX gene promoters included numerous biotic/abiotic-related and hormone-response elements [17]. It has been shown that WRKY transcription factors can regulate the expression of SPX members. For example, both WRKY6 and WRKY42 can regulate the transcription of the AtPHO1 gene by binding to the W-box element [30]. We identified a total of 18 MdSPX genes with W-box elements in their promoter regions, indicating that these genes may be regulated by WRKY transcription factor (Fig. 5; Table S3). P1BS (GNATATNC) is a known cis-acting element in response to Pi deficiency and clusters at the promoters of a lot of PSR genes [27, 31, 32]. For example, 18 ZmSPX genes contain P1BS elements in their promoter regions and 31 BnaSPX genes contain P1BS elements in their promoter regions [16, 18]. In our results, except for MdPHO1.9 and MdPHO1.7, the remaining 24 MdSPX genes contained different numbers of P1BS in their promoter regions (Fig. 5), predicting that the expression of these SPX genes in apple could be similarly induced by Pi deficiency. The position, number, and flanking sequence of all elements within a promoter region directly affects promoter activity. The enrichment of different promoter elements suggests that MdSPXs might be regulated by a series of related transcription factors.

Analysis of MdSPXs expression under different periods of low Pi treatment showed that several MdSPX genes were in response to Pi stress, with different expression patterns in the self-rooted apple stock superior ‘12 − 2’ (Fig. 7). Many studies have shown that some SPX subfamily genes are up-regulated under low Pi, for instance AtSPX and AtSPX2 [33], and we found that most MdSPX subfamily genes, except MdSPX1 and MdSPX5, were significantly induced in response to low Pi in apple, a result consistent with earlier studies [13, 34]. MdSPX2 and MdSPX3 were significantly expressed in both ‘12 − 2’ and ‘M9T337’ plants under low Pi stress condition, but the expression levels of MdSPX2 and MdSPX3 in ‘12 − 2’ under Pi deficient condition were significantly higher than that in ‘M9T337’, which further suggests the important role of the SPX genes in response to low Pi stress.

We also predicted the protein interaction networks of the 26 MdSPX protein sequences queried by STRING online software, which showed that multiple MdSPXs might interact with MdPHR1. AtPHR1 participates in Pi homeostasis by interacting with SPX structural domain-containing proteins at low Pi levels [33]. The results of the Y2H assay showed that MdSPX2, MdSPX3, MdSPX4, and MdSPX6 interacted with MdPHR1, while MdSPX1 and MdSPX5 did not interact with MdPHR1, a result similar to that of maize and rice. These findings suggest that a relatively conserved regulatory network for phosphorus stress response exists in self-rooted apple stock.


In the apple genome, 26 MdSPX genes were identified and divided into four subfamilies. To fully understand the biological features of these MdSPX genes, gene structures, conserved motifs, gene duplications, phylogenetic relationships and cis-elements were analysed in detail. The results of the expression of all 26 MdSPX genes at different times of Pi stress showed that some MdSPXs play key roles in Pi deficient response, especially MdSPX2, MdSPX3, and MdPHO1.5, which were highly responsive to low Pi stress. Some members might function under low Pi by interacting with MdPHR1. In conclusion, these results improved the understanding of the apple SPX gene family and contribute to future biological studies of MdSPX genes in self-rooted apple stock.


Identification of MdSPX genes in apple

MdSPX gene family members were identified by Blastp search in Malus×domestica reference genome database using 20 Arabidopsis SPX proteins as query sequences. Additionally, the Hidden Markov Model (HMM) SPX domain (PF03105) in the Pfam database ( were used to search for MdSPX genes. All candidate genes were then further verified using the Pfam and SMART ( Twenty-six MdSPX genes were finally identified in the apple genome and named in accordance with their phylogenetic relationship to Arabidopsis SPX genes.

Multiple sequence alignment and phylogenetic analysis

To investigate the evolutionary relationships of SPX proteins in Arabidopsis thaliana, Solanum lycopersicum, Zea mays, Oryza sativa, Malus domestica and Triticum aestivum, multiple alignments of 20 AtSPXs, 18 SlSPXs, 32 ZmSPXs, 15 OsSPXs, 26 MdSPXs, and 46 TaSPXs were performed by CLUSTALW, and the obtained results were applied to generate a phylogenetic tree via the neighbour-joining (NJ) method with 1000 bootstrap replicates in MEGA 7. The phylogenetic tree was landscaped using the EvolView tool ( It was then classified into four subfamilies based on SPX gene family characteristics.

Analysis of gene structure and conserved motifs

The DNA and cDNA sequences of all MdSPX and AtSPX genes were extracted from the Malus domestica and Arabidopsis genomes, respectively, and then the structures of the SPX genes were characterised using the Gene Structure Display Server (GSDS: The MEME motif discovery tool ( was used for identifying the conserved motif structures of all MdSPX and AtSPX amino acid sequences. Visualization of the results was performed using TBtools.

Physicochemical properties and chromosome location analysis

The 26 MdSPX protein sequences were examined using the online website ExPASy ( to predict their amino acid lengths, molecular weights and isoelectric points (pI) [35]. Chromosome position information of apple was downloaded from the GDR database ( The 26 MdSPX genes were mapped to their corresponding chromosomes using Mapchart 2.32 software.

Gene duplication analysis

Each of the MdSPX genes was mapped to the apple chromosome using TBtools, based on physical positional data from the apple genomic database. The Multiple Collinearity Scan toolkit (MCScanX) was used with default parameters to score gene duplication events [36, 37].

Identification of cis-elements in the promoters of the MdSPX genes

We extracted 2-kb sequences upstream of the transcription start site of the 26 MdSPX genes from the Malus domestica database using the TBtools. All extracted target sequences were then uploaded to PlantCARE ( and PlantPan3 databases ( for cis-element analysis. The obtained data were then preliminarily collated and visualised using Tbtools [38].

Plant material, pi stress treatments, and qRT-PCR analysis

The deep-rooted ‘12 − 2’ (hybrid seedlings of Ralls and Malus spectabilis) self-rooted apple stock and ‘M9T337’ stock, an apple dwarf rootstock were screened. The seedlings of M. spectabilis were obtained from the Beijing Botanical Garden (Beijing, China) and those of Ralls and ‘M9T337’ stock were obtained from Shandong Institute of Pomology. The roots of ‘12 − 2’ were used for qRT-PCR analysis. The growth conditions used were those described by [39]. Modified Hoagland medium was used in self-rooted apple stock hydroponic cultivation as described by [18]. An RNAprep pure plant kit (Vazyme, Nanjing, China) was used to isolate total RNA from the samples according to the producer’s instructions. Reverse transcription was then performed with a EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (Trans, Beijing, China). Applied Biosystems 7500 real-time PCR system (Applied Biosystems) was used to perform qRT-PCR reactions using UltraSYBR Mixture (with ROX I; Cwbiotech). The results were standardized with apple 18 S gene. Three biological replicates were used for each test. Table S4 showed the primers used in this study.

Prediction of protein association networks by STRING

The 26 MdSPX amino acid sequences were uploaded to the online STRING software (; version 11.5), with the organism selected as " Malus domestica “. The highest scoring proteins were selected to construct the network after BLAST analysis. The MdSPXs not interacting with other proteins were deleted. Functional annotations were manually copied from BLAST results. The proteins MdPHR1 (XP_008358479.1), MdPHR1-like (XP_008372382.1), MdBRE1 (XP_008374050.1), and MdBRE1-like (XP_008364561.1) were named based on BLAST results.

Measurement of the total pi concentration and physiological characteristics

To investigate the Pi sensitivity of ‘12 − 2’ and ‘M9T337’, we measured the root length, shoot height, and the number of adventitious root of the plants on the 21st day after Pi stress treatments. The chlorophyll content was quantified with a SPAD system [18]. The measurement of the total Pi concentration was performed as described by [40].

Yeast two-hybrid (Y2H) assays

The full-length MdPHR1 CDS was cloned into the bait vector pGBKT7 and the MdSPX1, MdSPX2, MdSPX3, MdSPX4, MdSPX5, and MdSPX6 CDSs were cloned into the prey vector pGADT7 to generate recombinant plasmid AD-MdSPX1, AD-MdSPX2, AD-MdSPX3, AD-MdSPX4, AD-MdSPX5, and AD-MdSPX6. Y2H assays were performed using yeast strain AH109 (Clontech) according to the manufacturer’s instructions. The yeast cells were plated on medium lacking Trp and Leu (SD/-Trp-Leu) and cultured at 28 °C. For interaction screening, the colonies were transferred into medium lacking Trp, Leu, His and adenine (SD/-Trp-Leu-His-Ade) with X-a-gal. Empty vector pGADT7 was used as negative controls.

Statistical analysis

In this study, the error bars represented the standard error (SE) from at least three biological replicates. The analysis of statistical significance was performed with the student’s t-test at P < 0.05 as described [41].

Data availability

The relevant materials are available from the corresponding authors on reasonable request. The links in this study for the data analyzed are as follows: Pfam database (; SMART (; EvolView tool (; Gene Structure Display Server (GSDS:; MEME motif discovery tool (; ExPASy (; GDR database (; PlantCARE (; PlantPan3 databases (; STRING software (; version 11.5).

Change history

  • 22 May 2024

    In the originally published articles the figures appeared out of order and Fig. 9 was monochrome. The article has been updated to rectify the errors.





Arabidopsis thaliana


Malus domestica


quantitative reverse transcription-PCR


Coding Sequence




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This work was supported by the National Natural Science Foundation of China (32102310 and 32202484), the Natural Science Foundation of Shandong Province (ZR2022QC008), the Shandong Key Research and Development Program, China (2021LZGC007 and 2022TZXD009). The funds played no role in study design, data analysis, and manuscript preparation.

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Authors and Affiliations



Z.W., Y.Y. and J.L. conceived and designed the experiments. Z.W., X.Z., X.Y., H.T., L.F. and J.L. performed the experiments. Z.W. analyzed the data and wrote the manuscript. Y.Y. and J.L. revised the manuscript. All authors have read and approved this manuscript.

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Correspondence to Yanlei Yin or Jialin Li.

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Additional file 1

Supplementary Fig. S1 to S2. (PDF 307 kb)

Additional file 2

Table S1 Grouping of SPX proteins. (XLSX 10.2 kb)

Additional file 3

Table S2 All tandem duplication and segmental duplication in apple genome. (XLSX 297 kb)

Additional file 4

Table S3Cis-element analysis of MdSPX genes. (XLSX 21.6 kb)

Additional file 5

Table S4 The primers used in this study. (XLSX 16.0 kb)

Supplementary Material 1

Supplementary Material 2

Supplementary Material 3

Supplementary Material 4

Supplementary Material 5

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Wang, Z., Zhang, X., Yang, X. et al. Evolution of the SPX gene family and its role in the response mechanism to low phosphorus stress in self-rooted apple stock. BMC Genomics 25, 488 (2024).

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