**3. Discussion**

The proteins encoded by SAP genes comprise large families and are broadly distributed in higher plants [2]. Apple is an economically important woody plant and the most widely cultivated fruit crop in the world. Sequencing of its genome has provided a good platform for genome-wide analyses of all putative gene families in apple, including the DREB [29], MYB [30], MADS-box [31], and WRKY [32,33] families. However, genome-wide information about apple SAP genes has remained unknown, while members of that family have been identified in other plant species [6,10,11,14,24,25]. Moreover, the content of SAP genes varies substantially among species. For example, *Brassica rapa*, *Glycine max*, *Solanum tuberosum*, *Salix purpurea*, *Populus trichocarpa*, and cotton each have a relatively large number

of SAP members, i.e., 28, 26, 19, 19, 19, and 19, respectively; while *Chlamydomonas reinhardtii*, *Lotus japonicus*, *Carica papaya*, and *Amborella trichopoda* have relatively few, i.e., 2, 6, 7, and 7, respectively (Table 3). Here, we determined that the apple genome contains 30 SAP genes, making this family much larger than in any other species.


**Table 3.** Numbers of SAP gene family members in various species.

Segmental, tandem, and whole-genome duplications are critical for both the diversification of gene functions and the rearrangement and expansion of genomes [31–34]. Whole-genome duplication events have occurred in apple [26], and tandem, segmental, and whole-genome duplications have caused some apple gene families to expand, including the MYB [30], MADS-box [31], and WRKY [32] families. We learned here that two *MdSAP* genes have undergone segmental duplication, and 17 have undergone tandem duplication. In addition, multiple gene pairs have each been linked to six potential chromosomal segmental duplications (Figure 5). Similar results have been reported for the Medicago SAP gene family. Our findings suggest that transposition events and the whole-genome and chromosomal segmental duplications have led to the expansion of the apple SAP gene family, and might partially explain why more SAP genes are present in apple than in any other species.

In several distinct species, the zinc finger types of some family members have either disappeared or increased in number. For example, the A20-A20-AN1 zinc finger occurs only in rice and *Eucalyptus grandis*; the A20 type is found in apple, rice, *Amborella trichopoda*, *B. rapa*, and grape (*Vitis vinifera*); the AN1-AN1-C2H2 zinc-finger exists in *Arabidopsis thaliana*, *A. lyrata*, *B. rapa*, *Capsella rubella*, *Thellungiella parvula*, and desert poplar (Table 3). We might speculate that the loss or the increase in zinc finger types of SAP genes in these genomes means that they are critical for the complicated enzymatic activity that is present in those species. In this study, we determined that apple SAPs are highly and structurally conserved based on analyses of gene structure, conserved domains, sequence alignments, 3D structures, and phylogenetics (Figures 1–4). Similar results have been reported for *Arabidopsis*, rice, maize, tomato, cotton, desert poplar, and medicago [6,10,11,14,24,25].

Although members of the SAP gene family in *Arabidopsis*, rice, tomato, and cotton are arranged into five groups [10,11,24], the SAP proteins expressed in *Arabidopsis*, desert poplar, *Populus trichocarpa*, *Salix purpurea*, and *S*. *suchowensis* are classified into two major groups: I (Ia–If) and II (IIa and IIb) [6]. Due to the small number of plant species that have been examined, we cannot yet accurately analyze the evolutionary relationships within that gene family. In this study, we were able to divide those members into two major groups (Ia–Id/IIa and IIb) by comparing the apple genome with SAP proteins from 31 other species (Figure 4). We believe that this result from our evolution analysis is convincing. Usually, exon–intron structural diversity can provide important evidence for phylogenetic relationships and play a valuable role in the evolution of gene families [31]. An intronless structure is typical of SAP genes in various species, and is a key characteristic of that family. However, one exception is the grape genome, for which only two VvSAP members lack introns, while 10 members each contain one intron. Furthermore, we discovered here that the intronless gene structure of SAP genes is the dominant arrangement among Group I members, whereas most of the Group II members contain at least one intron (Table 4 and Supplementary File B2). Thus, the prevalence of an intronless gene structure reflects the ancient origin of SAP genes, and links well with their rapid accumulation of transcripts due to reduced post-transcriptional processing [2,35].


**Table 4.** Statistics for numbers of intronless members within different groups of the SAP gene family.

Plant SAPs are quickly induced by multiple abiotic stresses [1,12–14,36–39]. They include rice *OsiSAP1*/*OsSAP1*, which responds to drought, salt, cold, submergence, mechanical wounding, and ABA [1]; and *ZFP177* (*OsSAP9*), which is also from rice, and shows enhanced expression in response to cold, heat, and PEG6000 [12]. The expression of *OsiSAP8* in tobacco and rice is enhanced by salt, cold, heat, desiccation, wounding, submergence, heavy metals, and ABA [36]. Similarly, SAP genes in *Aeluropus littoralis*, banana, *Arabidopsis*, and maize respond to salt, cold, drought, and osmotic stresses in a tissue and stress-specific manner [13,14,16,37–39]. In this study, we comprehensively analyzed the expression patterns of 13 cloned SAP genes under drought stress. Whereas the expression of *MdSAP15*, -*25*, and -*28* was significantly induced, transcripts levels for *MdSAP7* and -*21* mRNAs were significantly reduced (Figure 6B). Our results suggest that these genes have important roles in the response to water deficits.

The constitutive expression of SAP genes confers tolerance to multiple challenges. The overexpression of rice *OsiSAP1/OsSAP1*, *OsiSAP8*, and *AlSAP* in tobacco and rice increases their tolerance to numerous abiotic stresses [1,17,36,37,40]. Similar findings have been described for the overexpression of *AtSAP5* in cotton and *Arabidopsis* [13,41]. The overexpression of *AtSAP13* and *MusaSAP1* in *Arabidopsis* and banana leads to greater drought and salt tolerances [16,39]. The downregulation of *PagSAP1* improves salt tolerance in poplar and alters the regulation of genes involved in maintaining cellular ionic homeostasis [18]. We noted here that *MdSAP15* overexpression conferred increased tolerance to osmotic stress by increasing the root lengths and fresh weights of transgenic *Arabidopsis* seedlings when compared with the WT. This overexpression also influenced a

range of parameters associated with abiotic stress responses, including REL and the concentrations of chlorophyll, proline, and MDA, all of which are often used to evaluate the degree of plant tolerance under stress conditions [42–44]. Measured values for all of them were favorably affected in our transgenic lines (Figure 7). Finally, our experiments with induced water deficits demonstrated that the transgenic *Arabidopsis* plants showed milder stress symptoms when compared with the WT, and they also had higher survival rates during the period of rehydration and recovery from drought treatment (Figure 8). Taken together, these results indicate that the overexpression of *MdSAP15* in *Arabidopsis* plants leads to enhanced drought tolerance. This work provides a basis for exploring the molecular roles of SAPs and facilitates further investigations into the functions of these genes in abiotic stress responses. Our data also lay a solid foundation for future efforts to introduce improved apple cultivars.
