*4.3. The Sites of Apple PIP2 Expression*

Apple ESTs were retrieved through a BLASTN search of the GeneBank database, using as search terms each of the *MdPIP2* transcripts in turn. RNA was extracted from the roots of hydroponically-raised *M. hupehensis* seedlings (a triploid species characterized by facultative apomixis) which had formed 7–8 true leaves [39], and was processed for a series of qRT-PCR assays targeting seven *PIP2* genes. The relevant primers were designed using Beacon Designer 8 are shown in Table 3.



<sup>1</sup> Primers were designed to target the seven *MhPIP2* based on the sequences of their homologs in *M. domestica*.

## *4.4. Heterologous Expression of MpPIP2;1 in A. thaliana*

A full length copy of *MpPIP2;1* cDNA (JF834203.1) was PCR-amplified from an in-house p*MD19-T-MpPIP2;1* plasmid using a primer pair listed in Table 3 [41]. This was used to generate the construct p*CAMBIA2300-35S-MpPIP2;1*, which was introduced into *A. tumefaciens* strain GV3101, and from thence into *A. thaliana* (ecotype Col-0) using the floral dip method [42]. Selection for transgenic products was carried out by culturing on a medium containing 50 mg·L <sup>−</sup><sup>1</sup> kanamycin. The abundance of *MpPIP2;1* transcript produced in transgene homozygous T3 lines was evaluated using a qRT-PCR assay based on the primer pairs listed in Table 3.

## *4.5. Stress Tolerance Analysis*

For the purpose of assaying in vitro germination, surface-sterilized WT and transgenic seeds were laid on either solidified Murashige and Skoog (1962) medium (MS) [43], MS containing 0.25 M mannitol, or MS containing 0.15 M NaCl, and held for seven days under a 16 h photoperiod at 23 ◦C. Root elongation was assessed by culturing pre-germinated seedlings for two weeks on vertically oriented plants containing MS, MS + 0.25 M mannitol, or MS + 0.15 M NaCl. To test for both drought and salinity tolerances, seedlings were grown in pots containing equal amounts of soil after stratified and generated in soil for 3 weeks. The water loss rate was measured by weighing the detached leaves at different time points. The plants were then either subjected to drought by the withholding of water for 30 days, or salinity stress by irrigation with 0.3 M NaCl solution [44,45]. The assays for relative leaf water content, leaf electrolyte leakage, and MDA concentration have been described elsewhere. To assay for SOD, CAT, and POD activity and GSH content, leaves was homogenized in phosphate buffer (pH 7.5) and the resulting supernatants recovered after centrifugation were tested using commercially available kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The SOD Assay Kit was based on hydroxylamine method, the CAT Assay Kit was based on the method of ammonium molybdate, the POD Assay Kit was based on the hydrogen peroxide oxidation reaction, and the GSH assay kit is based on reduced glutathione reacting with 5,50 -dithiobis-2-nitrobenoic acid.
