*3.7. Expression of Ethylene Biosynthesis-, Cell Wall Modification- and Lignin Metabolism-Related Genes*

Expressions of several genes associated with ethylene biosynthesis were examined (Figure 4 and Table S6). In general, expression of *AdACS2* for the kiwifruit treated with preharvest chitosan at both concentrations was maintained at a relatively lower level than that of control during the storage (Figure 4a). This was apparent especially for the later stages of storage (i.e., 60 and 90 days). Another gene responsible for the biosynthesis of ethylene, *AdACO2*, displayed a similar pattern during the cold storage (Figure 4b). Preharvest treatment of chitosan at both concentrations suppressed expression of *AdACO2*, and this pattern of lower expression was observed until 60 days of cold storage.

**Figure 2.** Effects of preharvest chitosan application on (**a**) total phenolic content and (**b**) total lignin content during cold storage in 'Garmrok' kiwifruit. Vertical bars indicate SE with n = 3. \* indicates significant differences between treatments at each sampling date, according to the least significant difference (LSD) test at *p* ≤ 0.05.

**Figure 3.** Effects of preharvest chitosan application on (**a**) flesh firmness and (**b**) core firmness during cold storage in 'Garmrok' kiwifruit. Vertical bars indicate SE with n = 10. \* indicates significant differences between treatments at each sampling date, according to the least significant difference (LSD) test at *p* ≤ 0.05.

We examined several genes related to cell wall modification and the pattern of expression is shown in Figure 5 and Table S6. The preharvest treatment of chitosan suppressed the expression of *AdPGC* (Figure 5a) and *AdEXP1* (Figure 5b) at 30 and 60 days of the cold storage. The suppressed expression of these two genes was observed primarily with lower concentration of chitosan (100 mg·L−1). The effect of suppressed expression for *AdEXP2* (Figure 5c) was greater with a higher concentration of chitosan (500 mg·L<sup>−</sup>1).

**Figure 4.** Effects of preharvest chitosan application on relative expression of ethylene biosynthesisrelated genes (**a**) *AdACS2* and (**b**) *AdACO2* during cold storage in 'Garmrok' kiwifruit. Vertical bars indicate SE with n = 3. Actin gene was used as internal control and the quantitative RT-PCR data were analyzed using the 2−ΔΔCT method. \* Statistical difference caused by chitosan treatment was compared to untreated fruit (control) of each sampling date (*p* ≤ 0.05).

**Figure 5.** Effects of preharvest chitosan application on relative expression of cell wall-modification genes (**a**) *AdPGC*, (**b**) *AdEXP1*, and (**c**) *AdEXP2* during cold storage in 'Garmrok' kiwifruit. Vertical bars indicate SE with n = 3. Actin gene was used as internal control and the quantitative RT-PCR data were analyzed using the 2−ΔΔCT method. \* Statistical difference caused by chitosan treatment was compared to untreated fruit (control) of each sampling date (*p* ≤ 0.05).

As for genes associated with lignin metabolism, expression of *AcPAL*, a gene that is known to catalyze the first committed step in general phynylropanoid metabolism [32], was upregulated by the preharvest treatment of chitosan at both concentrations (Figure 6a and Table S6). The upregulation of *AcPAL* was most obvious at 30 days of cold storage. In addition, a gene considered to be an indicator of lignin biosynthesis [32], *AcCAD*, was upregulated at the end of cold storage (i.e., 90 days of storage) by both concentrations (Figure 6b and Table S6). A gene that is involved in the polymerization of monolignols to yield the lignin polymer [33], *AcPOD2*, was upregulated by the preharvest treatment of chitosan at both concentrations, especially toward the end of cold storage (Figure 6c and Table S6).

**Figure 6.** Effects of preharvest chitosan application on relative expression of lignin metabolismrelated genes (**a**) *AcPAL*, (**b**) *AcCAD*, and (**c**) *AcPOD2* during cold storage in 'Garmrok' kiwifruit. Vertical bars indicate SE with n = 3. Actin gene was used as internal control and the quantitative RT-PCR data were analyzed using the 2−ΔΔCT method. \* Statistical difference caused by chitosantreatment was compared to untreated fruit (control) of each sampling date (*p* ≤ 0.05).

#### **4. Discussion**
