*2.2. Phylogenetic Analysis*

The phylogenetic relationships between the set of MdAQPs with homologous proteins encoded by *A. thaliana*, poplar, and rubber (*Hevea brasiliensis*) is displayed in Figure 1. The analysis allowed the set of apple AQPs to be each assigned membership of one of the five plant AQP subfamilies, namely the MdPIPs (eleven members), the MdTIPs (thirteen members), the MdNIPs (eleven members), the MdSIPs (five members), and the MdXIPs (two members). The MdPIP members were further classified into the two subgroups, MdPIP1 and MdPIP2, the MdSIPs into the two subgroups, MdSIP1 and MdSIP2, and the MdTIPs into the five subgroups, MdTIP1-MdTIP5. The two MdXIPs belonged into the two subgroups MdXIP1 and MdXIP2, respectively, and the MdNIPs were divided into six subgroups, MdNIP1, 2, 4, 5, 6 and 7. On the basis of sharing a level of >90% similarity at the peptide level, 14 pairs of sequences were recognized, namely MdNIP1;1/MdNIP1;2, MdNIP2;1/MdNIP2;2, MdNIP5;1/MdNIP5;2, MdTIP1;3/MdTIP1;4, MdTIP4;1/MdTIP4;2, MdTIP2;1/MdTIP2;2, MdTIP3;1/MdTIP3;2, MdTIP5;1/MdTIP5;2, MdPIP1;1/MdPIP1;2, MdPIP2;1/MdPIP2;2, MdPIP2;3/MdPIP2;4, MdPIP2;6/MdPIP2;7, MdSIP1;2/MdSIP1;3, and MdSIP2;1/MdSIP2;2.

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 4 of 18

**Figure 1.** Phylogenetic tree of AQPs from *Arabidopsis thaliana*, *Populus trichocarpa*, *Hevea brasiliensis,* and *Malus domestica*. The protein sequences were aligned by ClustalX and the phylogenetic tree was constructed by the Neighbor-Joining method (1000 bootstrap replicates) in the MEGA6 software. The subgroups are marked by a colorful background(orange for TIPs, purple for PIPs, red for NIPs, blue for XIPs and gray for SIPs). **Figure 1.** Phylogenetic tree of AQPs from *Arabidopsis thaliana*, *Populus trichocarpa*, *Hevea brasiliensis,* and *Malus domestica*. The protein sequences were aligned by ClustalX and the phylogenetic tree was constructed by the Neighbor-Joining method (1000 bootstrap replicates) in the MEGA6 software. The subgroups are marked by a colorful background (orange for TIPs, purple for PIPs, red for NIPs, blue for XIPs and gray for SIPs).

#### *2.3. Chromosomal Location and Gene Structure 2.3. Chromosomal Location and Gene Structure*

It was possible to map 40 of the 42 *MdAQPs* on 16 of the 17 apple chromosomes, but neither *MdNIP1;2* nor *MdTIP3;2* could be placed (Figure 2). The sequence of each of the eleven *MdPIPs* featured three introns; all but one of the thirteen *MdTIPs* featured two introns (the exception was *MdTIP1;1* in which only one intron was present); eight of the eleven *MdNIP* sequences were interrupted by four introns, with three introns present in both *MdNIP5;1* and *MdNIP5;2*, and five in *MdNIP5;3*; three of the five *MdSIPs* harbored two introns, while neither *MdSIP1;2* nor *MdSIP1;3* featured any introns; finally, *MdXIP1;1* had one intron while *MdXIP2;1* included two introns (Figure 3). It was possible to map 40 of the 42 *MdAQPs* on 16 of the 17 apple chromosomes, but neither *MdNIP1;2* nor *MdTIP3;2* could be placed (Figure 2). The sequence of each of the eleven *MdPIPs* featured three introns; all but one of the thirteen *MdTIPs* featured two introns (the exception was *MdTIP1;1* in which only one intron was present); eight of the eleven *MdNIP* sequences were interrupted by four introns, with three introns present in both *MdNIP5;1* and *MdNIP5;2*, and five in *MdNIP5;3*; three of the five *MdSIPs* harbored two introns, while neither *MdSIP1;2* nor *MdSIP1;3* featured any introns; finally, *MdXIP1;1* had one intron while *MdXIP2;1* included two introns (Figure 3).

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 5 of 18

**Figure 2.** Distribution of *AQP* genes in apple chromosomes. Two genes (*MdNIP1;2* and *MdTIP3;2*) could not be localized on any chromosome. The scale is in megabases (Mb). **Figure 2.** Distribution of *AQP* genes in apple chromosomes. Two genes (*MdNIP1;2* and *MdTIP3;2*) could not be localized on any chromosome. The scale is in megabases (Mb).


*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 6 of 18

**Figure 3.** The exon-intron structure of apple *AQP* genes. Upstream/downstream region, exon, and intron are represented by blue box, yellow box, and grey line using GSDS software, respectively. **Figure 3.** The exon-intron structure of apple *AQP* genes. Upstream/downstream region, exon, and intron are represented by blue box, yellow box, and grey line using GSDS software, respectively.

#### *2.4. Conserved Residues in the Apple AQPs 2.4. Conserved Residues in the Apple AQPs*

The NPA motifs, ar/R filter, and Froger's positions were identified via a multiple alignment between the apple AQPs and other plant AQPs (Table 2). These conserved positions were critical for the substrate selectivity of AQPs. Both NPA domains were conserved in all MdPIP and MdTIP members, but the third residue of the first NPA in MdNIP5;1, MdNIP5;2, and MdNIP5;3 was serine rather than alanine, while in MdNIP5;1, MdNIP5;2, and MdNIP6;1, the third residue of the second NPA was valine rather than alanine; in MdNIP2;3 it was glutamate and in MdNIP5;3 it was isoleucine. The MdSIPs all carried a non-conserved third residue in the first NPA, while in addition, the first residue of the second NPA in MdSIP2;2 was serine rather than asparagine. Both the first The NPA motifs, ar/R filter, and Froger's positions were identified via a multiple alignment between the apple AQPs and other plant AQPs (Table 2). These conserved positions were critical for the substrate selectivity of AQPs. Both NPA domains were conserved in all MdPIP and MdTIP members, but the third residue of the first NPA in MdNIP5;1, MdNIP5;2, and MdNIP5;3 was serine rather than alanine, while in MdNIP5;1, MdNIP5;2, and MdNIP6;1, the third residue of the second NPA was valine rather than alanine; in MdNIP2;3 it was glutamate and in MdNIP5;3 it was isoleucine. The MdSIPs all carried a non-conserved third residue in the first NPA, while in addition, the first residue of the second NPA in MdSIP2;2 was serine rather than asparagine. Both the first and third residues of the first NPA of MdXIP1;1 were non-conserved and the third residues of the first NPA of MdXIP2;1 was valine. The ar/R

glycine-serine-glycine-arginine, alanine-isoleucine-glycine-arginine,

and third residues of the first NPA of MdXIP1;1 were non-conserved and the third residues of the first NPA of MdXIP2;1 was valine. The ar/R filter sequence was well conserved within each filter sequence was well conserved within each subfamily, but varied between the subfamilies. Each of the PIPs carried the conserved sequence phenylalanine-histidine-threonine-arginine. The greatest diversity for this motif was present among the NIPs, where each of the tetrapeptides tryptophan-valine-alanine-arginine, glycine-serine-glycine-arginine, alanine-isoleucine-glycine-arginine, threonine-isoleucine-alanine-arginine, and alanine-valine-glycine-arginine was represented. With respect to the Froger's positions, there was also high conservation within each subfamily, but variability between subfamilies. While the P1 position was the only variable residue within the PIP and XIP subfamily members, both the P1 and P2 positions varied among members of TIP and SIP subfamilies, and the P1, P2, and P5 positions were were all non-conserved for NIP subfamily members.


**Table 2.** NPA motifs, ar/R filter, and Froger's positons of apple AQPs.

NPA, Asparagine-Proline-Alanine; Ar/R, aromatic/arginine; LE, loop E; LB, Loop B; H2, transmembrane helix 2; H5, transmembrane helix 5.

*2.5. The Site of Apple PIP2 Expression* 

#### *2.5. The Site of Apple PIP2 Expression* a subgroup of the *M. domestica AQP* gene family, the products of which are likely important regulators of water transport across the plasma membrane. The distribution of these hits among

transmembrane helix 2; H5, transmembrane helix 5.

A search of the set of apple ESTs deposited in GenBank resulted in 685 hits for seven *MdPIP2s*, a subgroup of the *M. domestica AQP* gene family, the products of which are likely important regulators of water transport across the plasma membrane. The distribution of these hits among each *PIP2* gene and the organ are shown in Figure 4. The most well represented gene was *MdPIP2;4* (218 hits) and the least well represented was *MdPIP2;5* (seven hits). The site of transcription of these genes can be inferred from the frequency of their transcripts' occurrence in the 76 cDNA libraries assembled from various organs. In the bud libraries, there were 116 such ESTs out of a total of 54,099 sequences; there were 104 out of 54,120 in the leaf libraries; there were 73 out of 35,380 in the stem libraries; there was 94 out of 12,679 in the root libraries; there were 117 out of 44,772 in the flower libraries; there were 176 out of 104,341 in the fruit libraries; and finally out of libraries constructed from in vitro cultured cells, there were 5 out of 5652. When the abundance of transcripts generated from seven of the *PIP2* genes was evaluated by applying a quantitative real-time PCR (qRT-PCR) assay to RNA extracted from *M. hupehensis* root tissue, *PIP2;1* appeared to be the gene most strongly transcribed (Figure 5). Since both drought and salinity stress are sensed by roots, *PIP2;1* was chosen for a detailed functional analysis. In particular, the copy present in *Malus prunifolia* was selected, as this species provides a source of drought-tolerant rootstocks [22]. each *PIP2* gene and the organ are shown in Figure 4. The most well represented gene was *MdPIP2;4* (218 hits) and the least well represented was *MdPIP2;5* (seven hits). The site of transcription of these genes can be inferred from the frequency of their transcripts' occurrence in the 76 cDNA libraries assembled from various organs. In the bud libraries, there were 116 such ESTs out of a total of 54,099 sequences; there were 104 out of 54,120 in the leaf libraries; there were 73 out of 35,380 in the stem libraries; there was 94 out of 12,679 in the root libraries; there were 117 out of 44,772 in the flower libraries; there were 176 out of 104,341 in the fruit libraries; and finally out of libraries constructed from in vitro cultured cells, there were 5 out of 5652. When the abundance of transcripts generated from seven of the *PIP2* genes was evaluated by applying a quantitative real-time PCR (qRT-PCR) assay to RNA extracted from *M. hupehensis* root tissue, *PIP2;1* appeared to be the gene most strongly transcribed (Figure 5). Since both drought and salinity stress are sensed by roots, *PIP2;1* was chosen for a detailed functional analysis. In particular, the copy present in *Malus prunifolia* was selected, as this species provides a source of drought-tolerant rootstocks [22].

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 8 of 18

MdTIP2;3 NPA NPA H I G R T S A Y W

MdTIP3;1 NPA NPA H I A R T A A Y W

MdTIP3;2 NPA NPA H I A R T A A Y W

MdTIP4;1 NPA NPA H I A R S S A Y W

MdTIP4;2 NPA NPA H I A R S S A Y W

MdTIP5;1 NPA NPA N V G C T A A Y W

MdTIP5;2 NPA NPA N V G C I A A Y W

MdXIP1;1 SPV NPA V V V R M C A F W

MdXIP2;1 NPV NPA I T V R V C A F W NPA, Asparagine-Proline-Alanine; Ar/R, aromatic/arginine; LE, loop E; LB, Loop B; H2,

**Figure 4.** The frequency of the various *MdPIP2* ESTs present in cDNA libraries constructed from **Figure 4.** The frequency of the various *MdPIP2* ESTs present in cDNA libraries constructed from RNA extracted from buds, leaves, stems, roots, flowers, fruit and in vitro cultured cells.

RNA extracted from buds, leaves, stems, roots, flowers, fruit and in vitro cultured cells.

of three replicates.

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 9 of 18

**Figure 5.** Transcriptional profiling of seven *PIP2* genes in the root of *M. hupehensis*. RNA was extracted from the roots of hydroponically-raised *M. hupehensis* seedlings which had formed 7–8 **Figure 5.** Transcriptional profiling of seven *PIP2* genes in the root of *M. hupehensis*. RNA was extracted from the roots of hydroponically-raised *M. hupehensis* seedlings which had formed 7–8 true leaves. Values show in the form mean ± SD (*n* = 3). **Figure 5.** Transcriptional profiling of seven *PIP2* genes in the root of *M. hupehensis*. RNA was extracted from the roots of hydroponically-raised *M. hupehensis* seedlings which had formed 7–8 true leaves. Values show in the form mean ± SD (*n* = 3).

#### true leaves. Values show in the form mean ± SD (*n* = 3). *2.6. The Abiotic Stress Tolerance of A. thaliana Plants Heterologously Expressing MpPIP2;1 2.6. The Abiotic Stress Tolerance of A. thaliana Plants Heterologously Expressing MpPIP2;1*

of the three transgenic lines was then compared with that of wild type (WT) Col-0 plants.

*2.6. The Abiotic Stress Tolerance of A. thaliana Plants Heterologously Expressing MpPIP2;1*  A set of ten independent *A. thaliana* transgenics heterologously expressing *MpPIP2;1* was obtained, A set of ten independent *A. thaliana* transgenics heterologously expressing *MpPIP2;1* was obtained, and three of these were randomly selected to advance to the T3 generation (Figure 6). The performance of the three transgenic lines was then compared with that of wild type (WT) Col-0 plants. A set of ten independent *A. thaliana* transgenics heterologously expressing *MpPIP2;1* was obtained, and three of these were randomly selected to advance to the T3 generation (Figure 6). The performance

and three of these were randomly selected to advance to the T3 generation (Figure 6). The performance

**Figure 6.** Relative expression of *MpPIP2;1* in three *A. thaliana* transgenic lines heterologously **Figure 6.** Relative expression of *MpPIP2;1* in three *A. thaliana* transgenic lines heterologously expressing *MpPIP2;1* using qRT-PCR with Col-0 as control. The value presented was the mean ± SD **Figure 6.** Relative expression of *MpPIP2;1* in three *A. thaliana* transgenic lines heterologously expressing *MpPIP2;1* using qRT-PCR with Col-0 as control. The value presented was the mean ± SD of three replicates.

expressing *MpPIP2;1* using qRT-PCR with Col-0 as control. The value presented was the mean ± SD of three replicates. The contrasting effect of drought stress on the transgenic and WT plants is illustrated in Figure 7. Under well-watered conditions, the growth of the transgenic lines was indistinguishable from that of WT plants. However, when water was withheld for 30 days, none of the WT plants remained viable as they were unable to maintain a sufficient level of leaf hydration (their relative water content fell to 10.5%); in contrast, many of transgenic plants survived, maintaining a leaf relative water content of about 20%. The post-stress survival rate of these latter plants was between 10.3% and 23.5%. A comparison of the leaf malondialdehyde (MDA) content showed that less of this stress marker accumulated in the transgenic plants than in WT plants; similarly, it was established that the relative electrolyte leakage of leaves sampled from the transgenic plants was lower than that of WT The contrasting effect of drought stress on the transgenic and WT plants is illustrated in Figure 7. Under well-watered conditions, the growth of the transgenic lines was indistinguishable from that of WT plants. However, when water was withheld for 30 days, none of the WT plants remained viable as they were unable to maintain a sufficient level of leaf hydration (their relative water content fell to 10.5%); in contrast, many of transgenic plants survived, maintaining a leaf relative water content of about 20%. The post-stress survival rate of these latter plants was between 10.3% and 23.5%. A comparison of the leaf malondialdehyde (MDA) content showed that less of this stress marker accumulated in the transgenic plants than in WT plants; similarly, it was established that the relative electrolyte leakage of leaves sampled from the transgenic plants was lower than that of WT leaves. The activity of each of the enzymes superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), as well as the content of glutathione (GSH), were all greater in the transgenic The contrasting effect of drought stress on the transgenic and WT plants is illustrated in Figure 7. Under well-watered conditions, the growth of the transgenic lines was indistinguishable from that of WT plants. However, when water was withheld for 30 days, none of the WT plants remained viable as they were unable to maintain a sufficient level of leaf hydration (their relative water content fell to 10.5%); in contrast, many of transgenic plants survived, maintaining a leaf relative water content of about 20%. The post-stress survival rate of these latter plants was between 10.3% and 23.5%. A comparison of the leaf malondialdehyde (MDA) content showed that less of this stress marker accumulated in the transgenic plants than in WT plants; similarly, it was established that the relative electrolyte leakage of leaves sampled from the transgenic plants was lower than that of WT leaves. The activity of each of the enzymes superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), as well as the content of glutathione (GSH), were all greater in the transgenic plants than in the WT ones. Consistent with this result, the detached leaves of transgenic lines lost water slower than that of WT leaves. After 5 h of dehydration, the water loss rate for transgenic lines OE1, OE2, and OE3 were 17.6%, 8.8%, and 4.13% lower than that of WT, respectively.

leaves. The activity of each of the enzymes superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), as well as the content of glutathione (GSH), were all greater in the transgenic plants than in the WT ones. Consistent with this result, the detached leaves of transgenic lines lost

plants than in the WT ones. Consistent with this result, the detached leaves of transgenic lines lost water slower than that of WT leaves. After 5 h of dehydration, the water loss rate for transgenic

lines OE1, OE2, and OE3 were 17.6%, 8.8%, and 4.13% lower than that of WT, respectively.

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 10 of 18

**Figure 7.** Heterologous expressing *MpPIP2;1* enhanced drought tolerance in *A. thaliana*. (**a**) Phenotypes of transgenic lines and wild type plants under drought stress; (**b**) survival rate after 30 days withholding of water and 7 days after rewatering; (**c**) relative water content after 30 days withholding of water; (**d**) water loss rate of detached leaves; (**e**) malondialdehyde (MDA) content; (**f**) relative electrolyte leakage; (**g**) superoxide dismutase (SOD) activity; (**h**) catalase (CAT) activity; (**i**) peroxidase (POD) activity; and (**j**) glutathione (GSH) content at 0, 15, and 30 days after water withheld. The value presented was the mean ± SD of three replicates, and the bar with different letter was significantly different between plants at *p* < 0.05. **Figure 7.** Heterologous expressing *MpPIP2;1* enhanced drought tolerance in *A. thaliana*. (**a**) Phenotypes of transgenic lines and wild type plants under drought stress; (**b**) survival rate after 30 days withholding of water and 7 days after rewatering; (**c**) relative water content after 30 days withholding of water; (**d**) water loss rate of detached leaves; (**e**) malondialdehyde (MDA) content; (**f**) relative electrolyte leakage; (**g**) superoxide dismutase (SOD) activity; (**h**) catalase (CAT) activity; (**i**) peroxidase (POD) activity; and (**j**) glutathione (GSH) content at 0, 15, and 30 days after water withheld. The value presented was the mean ± SD of three replicates, and the bar with different letter was significantly different between plants at *p* < 0.05.

A series of experiments were conducted to establish whether the constitutive expression of *MpPIP2;1* in *A. thaliana* had any effect on the level of tolerance to salinity stress, as imposed by exposure to 0.3 M NaCl for 14 days (Figure 8). The transgenic plants maintained a superior leaf hydration status compared to the WT plants: Their respective relative water contents were >80% and 49%. While the survival rate of WT plants was 46.4%, that of the transgenic plants was >90%. Compared to WT leaves, those of the transgenic plants accumulated less MDA, developed a lower relative electrolyte leakage, exhibited a higher activity of SOD, POD, and CAT, and their GSH content was greater. A series of experiments were conducted to establish whether the constitutive expression of *MpPIP2;1* in *A. thaliana* had any effect on the level of tolerance to salinity stress, as imposed by exposure to 0.3 M NaCl for 14 days (Figure 8). The transgenic plants maintained a superior leaf hydration status compared to the WT plants: Their respective relative water contents were >80% and 49%. While the survival rate of WT plants was 46.4%, that of the transgenic plants was >90%. Compared to WT leaves, those of the transgenic plants accumulated less MDA, developed a lower relative electrolyte leakage, exhibited a higher activity of SOD, POD, and CAT, and their GSH content was greater.

**Figure 8.** Heterologous expressing *MpPIP2;1* enhanced salt tolerance in *A. thaliana*. (**a**) Phenotypes of transgenic lines and wild type plants treated with 0.3 M NaCl for 14 days; (**b**) Survival rate and (**c**) relative water content after exposure to NaCl; (**d**) MDA content; (**e**) relative electrolyte leakage; (**f**) SOD activity; (**g**) CAT activity; (**h**) POD activity; and (**i**) GSH content at 0 and 14 days after NaCl treatment. The value presented was the mean ± SD of three replicates, and the bar with different letter was significantly different between plants at *p* < 0.05. **Figure 8.** Heterologous expressing *MpPIP2;1* enhanced salt tolerance in *A. thaliana*. (**a**) Phenotypes of transgenic lines and wild type plants treated with 0.3 M NaCl for 14 days; (**b**) Survival rate and (**c**) relative water content after exposure to NaCl; (**d**) MDA content; (**e**) relative electrolyte leakage; (**f**) SOD activity; (**g**) CAT activity; (**h**) POD activity; and (**i**) GSH content at 0 and 14 days after NaCl treatment. The value presented was the mean ± SD of three replicates, and the bar with different letter was significantly different between plants at *p* < 0.05.

#### *2.7. Germination and Root Elongation of MpPIP2;1 Transgenics Exposed to Either Salinity or Osmotic 2.7. Germination and Root Elongation of MpPIP2;1 Transgenics Exposed to Either Salinity or Osmotic Stress*

*Stress*  An experiment was conducted to establish whether the constitutive expression of *MpPIP2;1* in *A. thaliana* had any effect on germination and/or root elongation in the presence of either salinity or osmotic stress (Figure 9). When the seed was imbibed in the absence of a stress agent (mannitol or NaCl), the rate of germination of both the WT and transgenic seed was high, and there were no significant differences between the germination rates of WT and transgenic seeds. However, in the presence of either 0.25 M mannitol or 0.15 M NaCl, the rate of germination of the WT seeds fell to An experiment was conducted to establish whether the constitutive expression of *MpPIP2;1* in *A. thaliana* had any effect on germination and/or root elongation in the presence of either salinity or osmotic stress (Figure 9). When the seed was imbibed in the absence of a stress agent (mannitol or NaCl), the rate of germination of both the WT and transgenic seed was high, and there were no significant differences between the germination rates of WT and transgenic seeds. However, in the presence of either 0.25 M mannitol or 0.15 M NaCl, the rate of germination of the WT seeds fell to just 10%, while that of the transgenic seeds remained >60%. Similarly, the ability of roots to elongate was

just 10%, while that of the transgenic seeds remained >60%. Similarly, the ability of roots to elongate

the same for the WT and transgenic seedlings under non-stressful conditions, but the extent of its inhibition by the presence of either mannitol or NaCl differed between the transgenic and WT seedlings. was the same for the WT and transgenic seedlings under non-stressful conditions, but the extent of its inhibition by the presence of either mannitol or NaCl differed between the transgenic and WT seedlings.

**Figure 9.** Heterologous expressing *MpPIP2;1* enhanced seeds germination and root elongation in *A. thaliana* either under salinity or osmotic stress. (**a**) The phenotype and (**b**) the statistical analyses of the root lengths of transgenic lines and wild type seedlings growing on MS medium (CK), or MS medium with 0.15 M NaCl or 0.25 M mannitol for 14 days; (**c**) germination rate of transgenic and wild type seeds on different mediums for 7 days. The value presented was the mean ± SD of three replicates, and the bar with different letter was significantly different between plants at *p* < 0.05. **Figure 9.** Heterologous expressing *MpPIP2;1* enhanced seeds germination and root elongation in *A. thaliana* either under salinity or osmotic stress. (**a**) The phenotype and (**b**) the statistical analyses of the root lengths of transgenic lines and wild type seedlings growing on MS medium (CK), or MS medium with 0.15 M NaCl or 0.25 M mannitol for 14 days; (**c**) germination rate of transgenic and wild type seeds on different mediums for 7 days. The value presented was the mean ± SD of three replicates, and the bar with different letter was significantly different between plants at *p* < 0.05.

#### **3. Discussion 3. Discussion**

tolerance [27].

The systematic scanning of the content of AQP-encoding genes in the apple genome reported here resulted in the identification of 42 such genes. AQPs make an important contribution to the way in which plants control their uptake of water, and hence represent a key component of their response to drought and osmotic stress [23]. Thus, gaining a full understanding of how apple plants regulate their water balance and adapt to drought and osmotic stress will likely involve revealing the function of many of this set of genes. There is already some experimental evidence which supports the participation of *AQPs* in the stress response of apple. According to Hu et al. (2003), the transcription of both *MdPIP1a* and *MdPIP1b* (here renamed as, respectively, *MdPIP1;2* and *MdPIP1;1*) are up-regulated by osmotic stress [24]. The *M. zumi* homolog of *MdPIP1;1* has been shown to be inducible by salinity (as well as by low temperature) stress [25]. Meanwhile, the constitutive expression of *MzPIP2;1* (homolog of *MdPIP2;4*) in *A. thaliana* has a positive effect on drought tolerance and a small positive one on salinity tolerance [26], and the expression of *MzPIP1;3* (homolog of *MdPIP1;3*) in tomato has been shown to enhance the plants' drought The systematic scanning of the content of AQP-encoding genes in the apple genome reported here resulted in the identification of 42 such genes. AQPs make an important contribution to the way in which plants control their uptake of water, and hence represent a key component of their response to drought and osmotic stress [23]. Thus, gaining a full understanding of how apple plants regulate their water balance and adapt to drought and osmotic stress will likely involve revealing the function of many of this set of genes. There is already some experimental evidence which supports the participation of *AQPs* in the stress response of apple. According to Hu et al. (2003), the transcription of both *MdPIP1a* and *MdPIP1b* (here renamed as, respectively, *MdPIP1;2* and *MdPIP1;1*) are up-regulated by osmotic stress [24]. The *M. zumi* homolog of *MdPIP1;1* has been shown to be inducible by salinity (as well as by low temperature) stress [25]. Meanwhile, the constitutive expression of *MzPIP2;1* (homolog of *MdPIP2;4*) in *A. thaliana* has a positive effect on drought tolerance and a small positive one on salinity tolerance [26], and the expression of *MzPIP1;3* (homolog of *MdPIP1;3*) in tomato has been shown to enhance the plants' drought tolerance [27].

Both moisture and nutrient stress are initially sensed by a plant's roots. Based on its relatively high transcript abundance (inferred both indirectly from the frequency of its representation in EST libraries and directly through a qRT-PCR analysis), *PIP2;1* is the *PIP2* gene most strongly transcribed in the roots of the apple plant. This same gene has been shown, using a suppression subtractive hybridization method, to be up-regulated in response to moisture deficit [28], while two *MdPIP2;1* ESTs have been identified in cDNA libraries developed from the roots of plants from which water had been withheld for a week (LIBEST\_024527). Thus the evidence points to the conclusion that the product of *PIP2;1* is an important component of the apple plant's response to moisture stress. This evidence has been strengthened by the demonstration here that heterologously expressing the gene in *A. thaliana* had a positive effect on the plant's tolerance of both drought and salinity.

A commonly observed plant response to abiotic stress is to accumulate reactive oxygen species (ROS), which become cytotoxic when present in excess [29]. When *A. thaliana* plants heterologously expressing *MpPIP2;1* were exposed to either drought or salinity stress, both the MDA content and the relative electrolyte leakage of their leaves were below the levels shown by WT leaves; both of these traits are correlated with ROS-mediated cellular damage [30]. The maintenance of a non-damaging level of cellular ROS content is achieved both by the activity of a number of enzymes and the synthesis of antioxidant compounds [31]. Both the activity of the enzymes SOD, CAT, and POD and the content of the antioxidant compound GSH were higher in the transgenic than in the WT *A. thaliana* plants subjected to stress. The conclusion is that the product of *MpPIP2;1* likely contributes to protecting the transgenic plants experiencing drought stress by enhancing their ability to control the accumulation of ROS. Similar conclusions have been drawn with respect to AQPs in a number of plant systems [32], so it is reasonable to propose that the product of *MpPIP2;1*, a gene which is strongly transcribed in the root of *M. prunifolia*, is an important determinant of the drought stress response when expressed in its native context.

#### **4. Materials and Methods**

#### *4.1. Identification of the Set of AQP Genes in the Apple Genome*

Apple AQP sequences were recovered from the NCBI Protein database (www.ncbi.nlm.nih.gov/ protein) by entering as a keyword search "(aquaporin OR MIP) AND Malus". The resulting hits were confirmed as genuine AQPs by submitting them to the NCBI Conserved Domain database (www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The sequences of identified apple AQPs were used as queries to search Malus x domestica Whole Genome v1.0 and GDDH13 Whole Genome v1.1 sequence for additional members with an E value less than 0.01. A phylogenetic analysis based on the deduced peptide sequences of AQPs encoded by *A. thaliana*, poplar, and rubber [33] was used assign the apple sequences to the five established AQP subfamilies. Multiple sequence alignments were carried out using ClustalX software [34], and an unrooted phylogenetic tree was constructed using MEGA6 software [35], applying the Neighbor-Joining method and 1000 bootstrap replicates.

#### *4.2. Chromosomal Location, Gene Structure, and Protein Properties of Apple AQPs*

The GDDH13 assembly was used to reveal the chromosomal location for each of the *MdAQPs* and to determine the intron/exon structure of each gene. The latter was visualized using GSDS software (bio.tools/GSDS) [36]. The pI and molecular weight of the deduced AQPs were predicted using the ExPASY program (web.expasy.org/compute\_pi/). Transmembrane regions were detected using TMHMM software (www.cbs.dtu.dk/services/TMHMM/) [37], and subcellular localizations were predicted using WoLF PSOR software (wolfpsort.hgc.jp/) [38]. Sequences representing conserved domains, NPA motifs, the ar/R filter, and the Froger positions were manually identified, based on multiple sequence alignments of apple AQPs with heterologous AQPs.
