The test plant materials were obtained from 12-year-old “Royal Gala” apple (Malus domestica Borkh. “Royal Gala”) trees cultivated at the Shandong Agricultural University experimental station and from 1-month-old self-rooted apple seedlings [Malus hupehensis (Pamp.) Rehd. “pinyiensis.”] cultivated in the laboratory of molecular biology of fruit trees of the Academy of Horticultural Science and Engineering, Shandong Agricultural University. After taking the roots, stems, leaves, flowers (initial flowering), peels, and pulps of “Royal Gala,” the samples were quickly added in liquid nitrogen and timely transferred to an ultra-low temperature refrigerator for storage for subsequent test operations.

The apple shoot cultures, derived from “Royal Gala,” were stored at 24°C on MS solid medium (containing 0.8% agar, 0.2 mg/L NAA, 0.5 mg/L 6-BA, and 0.1 mg/L GA3) under a photoperiod (8/16 h dark/light) for 25 days. To obtain self-rooted plantlets, the 4-week-old shoot cultures were transferred to a root-inducing MS solid medium containing 0.2 mg/L IAA. For gene expression analysis, 25-day-old self-rooted apple seedlings were treated with 150 mM NaCl, 10% PEG 6000, and 100 μM ABA. Apple seedlings were sampled at different time points after treating for 0, 1, 3, 6, and 12 h and were quickly placed in liquid nitrogen and stored in an ultra-low temperature refrigerator for subsequent experiments (Yamaguchi-Shinozaki and Shinozaki 1994).

“Orin” apple calli were grown on MS solid medium supplemented with 0.8% agar, 0.4 mg/L 6-BA, and 1.5 mg/L 2, 4-dichlorophenoxyacetic acid (2,4-D) at 25°C in the dark. After 15 days of growth, calli (WT and MdMSI1-1-OX) were transferred to the MS solid medium supplemented with 0 or 100 mM NaCl and placed in the dark for 16 days. Arabidopsis thaliana (Columbia) seeds were germinated on the MS solid medium at 22°C in a photoperiod (16 h/8 h, light/dark). The 3-day-old Arabidopsis seedlings (WT and MdMSI1-1-OE) were transferred to the MS solid medium that supplied with 0 or 100 mM NaCl for 14 days. After treatment, we measured the MDA content of calli following the study by (Ma et al., 2017) and the relative electronic conductivity level of Arabidopsis following the study by (Hu et al., 2013).

The full-length cDNA of MdMSI1-1 was cloned into the PRI-101 (35S promoter, GFP) vector to obtain its overexpression plasmid. The primer pairs MdMSI1-1-F (5′-ATGGGCAAAGAC -3′)/−R and (5′- AGGCTTTGCCGGTTC -3′) were used to amplify the full-length MdMSI1-1.

Apple calli were infected by Agrobacterium-mediated transformation to obtain MdMSI1-1 overexpression transgenic apple calli (MdMSI1-1-OX) (An et al., 2016). The transgenic Arabidopsis (MdMSI1-1-OE) were obtained by using the floral dip transformation method (Yang et al., 2021).

The apple “GDDH13” reference genome is from the Apple Genome and Epigenome database (GDDH13_1-1, https://iris.angers.inra.fr/gddh13/) (Daccord et al., 2017). Arabidopsis MSI proteins are from the NCBI (https://www.ncbi.nlm.nih.gov/). The apple MSIs were searched by using AtMSI sequences (BLASTp). In addition, apple MSIs were obtained by the HMM method with the E-value (10e⁻⁵) (http://pfam.xfam.org/) (Finn et al., 2014). The domains (CAF1C_H4-bd, WD40) were analyzed and confirmed by online software SMART (http://smart.embl-heidelberg.de/). The members of apple MSIs were finally obtained. The number of amino acids, molecular weight, isoelectric point, and other physical and chemical properties of MdMSI gene family members were analyzed by using ProtParam in online software EXPASY (https://web.expasy.org/protparam/). The subcellular localization of MdMSIs was predicted by online software WoLF PSORT (https://www.genscript.com/wolf-psort.html) (Horton et al., 2007).

MdMSI protein sequence alignment was performed by online software Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and visualized by Jalview (Jalview 2.11.1.4).

MdMSI protein sequences were downloaded from the GDDH13 website, AtMSIs from the TAIR (http://www.Arabidopsis.org/), and OsMSIs from the Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html). All MSI protein sequences of the apple, Arabidopsis, and rice were constructed using a phylogenetic tree by software Mega7 (Kumar et al., 2016). The phylogenetic tree was constructed by using the NJ method (Bootstrap set to 1,000), and 10 conserved motifs of MdMSIs and AtMSIs were analyzed by online software MEME (http://meme-suite.org/) (Bailey et al., 2006).

The secondary structures of MdMSI proteins were predicted by online software SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html). The tertiary structures of MdMSI proteins were predicted by homology modeling of online software Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi? id=index) (Kelley et al., 2015).

The interaction of AtMSI member-related proteins was predicted by online software STRING (https://stringdb.org) (Szklarczyk et al., 2019), and then, the related homologous apple MSI members were matched.

The chromosome position of apple MSI family members was visually analyzed by online software MG2C (http://mg2c.iask.in/mg2c_v2.1/). Using gene annotation information, the gene structure of MSI family members was analyzed by online software GSDS 2.0 (http://gsds.gao-lab.org/) (Hu et al., 2015).

The promoter region of about 2000 bp upstream of the apple MSI gene was submitted to the online software PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (Lescot et al., 2002) for cis-acting element analysis.

RNA was extracted from the apple material using the RNA plant plus Kit (Tiangen, Beijing), cDNA was obtained by reverse transcription of the extracted RNA using the primescript RT reagent kit a with gDNA eraser (Takara, Dalian), qRT-PCR was performed on an iCycler iQ5 system (Bio-RAD) instrument, and data on relative gene expressions were analyzed by 2^−ΔΔCT methods. The quantitative primer design for the MSI genes in the apple was performed by using online software Primer3Plus (http://primer3plus.com/cgi-bin/dev/primer3plus.cgi) (Untergasser et al., 2007), as shown in Supplementary Table S1.

We performed data significance analysis by DPS software. According to the Tukey-Kramer test, it indicated that different lowercase letters represent significant differences. Each experiment was repeated at least three times.

After stringent dereplication screening using the online software SMART and Pfam with AtMSIs as the query sequence, eight MdMSI members were finally identified from the “Golden Delicious” apple genome sequence, which were sequentially named MdMSI1-1 (MD02G1228600), MdMSI1-2 (MD07G1083900), MdMSI2 (MD15G1206400), MdMSI3-1 (MD05G1121700), MdMSI3-2 (MD10G1124300), MdMSI4-1 (MD02G1079600), MdMSI4-2 (MD09G1193500), and MdMSI4-3 (MD15G1207100) based on their homology to Arabidopsis MSI family genes. Using the genome annotation file and protein analysis website, the gene length, coding sequence, amino acid number, molecular weight, isoelectric point (pI), and subcellular localization of MdMSI genes were statistically analyzed. It was found that the length of MdMSI genes ranged from 2,673 to 5,972 bp: the shortest was MdMSI3-2 and the longest was MdMSI4-2. The length of the coding sequence ranged from 1,080 to 1,557 bp: the shortest was MdMSI3-2 and the longest was MdMSI4-1. The number range of amino acids was 359–518, the molecular weight was 40,171.82–57,353.08 Da, the isoelectric point was 4.60–6.08, and the pI was less than 7. By using online software WoLF PSORT, the subcellular localization of MdMSIs was predicted to be all in the nucleus (Table 1).

In the process of protein domain query by online software SMART, it was found that the eight MSI proteins of apple and the five MSI proteins of Arabidopsis contain two typical domains: one was the CAF1C_H4-bd domain, and the other was a typical WD40 domain (Figure 1). The MSI protein sequences of Arabidopsis and apple were compared by online software Clustal Omega and visualized by using the software tool Jalview 2.11.1.4. Arabidopsis and apple MSI proteins had only one highly conserved CAF1C_H4-bd domain. Another WD40 domain was composed of about 40 amino acids.

The phylogenetic tree was constructed by using apple, Arabidopsis, and rice MSIs, and the apple MSI was grouped according to the classification of Arabidopsis MSI. MSI were divided into three categories, MdMSI1-1 and MdMSI1-2 were divided into one category (I); MdMSI2, MdMSI3-1, and MdMSI3-2 were divided into one category (II); and MdMSI4-1, MdMSI4-2, and MdMSI4-3 were divided into one category (III) (Figure 2).

Using the online software MEME, we predicted 10 motifs in the MdMSI and AtMSI proteins (Figure 3). The motif distribution of MSI proteins was similar. All MSI proteins contain motifs 1, 5, and 6. However, there are also differences in the composition of MSI protein motifs in different groups of the evolutionary tree (Figure 2). For example, motif 7 was detected only in members of (III) (MdMSI4-1, MdMSI4-2, and MdMSI4-3 in apple and AtMSI4 and AtMSI5 in Arabidopsis).

The secondary structure of MdMSI protein was compared. The largest proportion of the secondary structure of eight proteins was random coil, followed by α-helix, and the smallest proportion was β-turn. Moreover, these proteins had roughly similar proportions of the four secondary structure elements (Table 2). The online software Phyre2 was used for the homology model to predict MdMSI families, and the results showed that the tertiary structure of the protein-conserved region of the MdMSI family was very consistent (Figure 4). The special structure was often related to its function, indicating that their function was very likely to be similar.

By using online software STRING, the protein network and potential function of the MdMSI protein interaction were predicted. MdMSI1-1 and MdMSI1-2 correspond to AtMSI1; MdMSI2 corresponds to AtMSI2; MdMSI3-1 and MdMSI3-2 correspond to AtMSI3; and MdMSI4-1, MdMSI4-2, and MdMSI4-3 correspond to AtFVE (Figure 5). According to the predicted results of the Arabidopsis MSI protein interaction network, AtMSI was associated with FIE protein, SWN protein, VRN2 protein, RNR1 protein, PCNA1 protein, and PCNA2 protein. In addition to interacting with reproductive development-related proteins, such as the FIE protein, SWN protein, and VRN2 protein, it was also associated with stress-related proteins, such as chloroplast posttranscriptional regulation-related protein RNR1 and PCNA1/2 protein interaction, which played an important role in DNA damage response. It showed that the MSI protein was regulated by multiple protein interactions.

Chromosome location and gene structure analysis of MdMSI genes showed that they were unevenly distributed on six chromosomes, of which only one gene was distributed on chr05, chr07, chr09, and chr10, which were MdMSI3-1, MdMSI1-2, MdMSI4-2, and MdMSI3-2 respectively. There were two genes on chr02 and chr15, respectively, MdMSI1-1 and MdMSI4-1, MdMSI2 and MdMSI4-3 (Figure 6A). The online software GSDS 2.0 was used to visually analyze the gene structure. The number of exons of the gene family was 6–15, and the number of introns was 5–14. It was very interesting that MdMSI1-1 and MdMSI1-2, MdMSI3-1, and MdMSI3-2 corresponding to AtMSI1 and AtMSI3, respectively, had six exons and five introns (Figure 6B). According to the analysis of the aforementioned apple MSI phylogenetic tree, it was speculated that the two groups of genes were likely to have similar functions. In addition, the three genes corresponding to AtMSI4 also had the same exon and intron, and their numbers are 15 and 14, respectively.

The promoter of MSI genes in apples was analyzed (Table 3). It was detected that there were a large number of cis-acting elements in response to hormones and stress, such as the ABRE element in response to abscisic acid, CGTCA of jasmonic acid, ERE of ethylene, P-box and GARE motif of gibberellin, TCA of salicylic acid, and TGA of auxin, and were in response to hypoxia, MBS in drought, TC-rich repeats of defense and stress, etc. In conclusion, it was speculated that apple MSI may play an important role in abiotic stress.

In order to explore the temporal and spatial expression of MdMSIs in apple, they were analyzed by qRT PCR in roots, stems, leaves, flowers, peels, and pulps. MdMSIs were expressed in all tested tissues (Figure 7). MdMSI1-1, MdMSI2, MdMSI3-1, and MdMSI4-1 were the most expressed in roots. The expression of MdMSI1-1 in roots was about two times higher than that in other tissues. Moreover, MdMSI1-2, MdMSI3-2, and MdMSI4-3 were highly expressed in stems and peels. They all had a low expression level in the leaf.

In order to study the response of apple MSI genes to abiotic stress, the seedlings of apple were treated with salt and drought. After salt stress, the relative expression levels of the other seven genes reached the maximum at 12 h, except MdMSI3-2 (Figure 8A). In addition, it was very interesting that the relative expression of the MdMSI gene family decreased first and then increased after salt stress treatment, except MdMSI3-1 and MdMSI4-1. After drought treatment, except MdMSI3-2 and MdMSI4-2, the overall expression of the other six genes in the MdMSI gene family showed an upward trend (Figure 8B). In addition, many cis-elements related to hormones were found in the analysis of the apple MSI promoter. Among them, the number and range of ABA were the most extensive in the MdMSI gene family. Therefore, the seedlings of apple were also treated with ABA. The expressions of MdMSI1-1, MdMSI1-2, MdMSI3-1, MdMSI3-2, MdMSI4-2, and MdMSI4-3 were similar, and they first showed an upward trend, peaked at 6 h, and then decreased (Figure 8C). The expression of MdMSI4-1 reached the highest levels at 3 h.

Among the MdMSIs promoters, a large number of cis-elements related to hormones and abiotic stress were identified in MdMSIs promoters, indicating that the family genes may be involved in abiotic stress response (Table 3). There were a lot of abiotic stress-related elements in the MdMSI1-1 promoter sequence, and its expression level was significantly affected by salt stress. Therefore, we predicted that it was very likely to participate in salt stress. We obtained three transgenic apple callus lines of overexpressed MdMSI1-1 (MdMSI1-1-OX1, MdMSI1-1-OX2, and MdMSI1-1-OX3) (Figure 9D). Under the control condition, there was no significant difference in fresh weight and MDA between WT and MdMSI1-1-OX, but under the salt treatment condition, the fresh weight of MdMSI1-1-OX was significantly lower than WT, and MDA was significantly higher than WT (Figures 9A–C). The results showed that MdMSI1-1 negatively regulated salt stress in apple.

In order to further verify the function of MdMSI1-1 in salt stress, we obtained three MdMSI1-1 overexpressions in Arabidopsis (OE1, OE2, and OE3) (Figure 10B). We treated 3-day-old Arabidopsis seedlings (WT and MdMSI1-1-OE) with 100 mM NaCl and found that MdMSI1-1-OE had lighter fresh weight and shorter primary root length than WT (Figures 10A,C,D). Moreover, we detected the relative electronic conductivity in Arabidopsis (Figure 10E). The relative electronic conductivity of MdMSI1-1-OE was higher than that of WT. These results showed that MdMSI1-1 increased the sensitivity of apple calli and Arabidopsis under salt stress.