*2.8. Expression Patterns of CaChi-Genes in Different Tissues*

To further elucidate the expression characteristics of CaChi genes in various vegetative and reproductive tissues (leaf, stem, root, seven developmental phases of placenta and pericarp), we carried out in silico analysis using public transcriptomic database of pepper [39,40]. The different expression patterns of CaChi in pepper exhibited higher variance in distinctive tissues and stages, fluctuation.

as demonstrated in heat map where from green to red display the index of expression (Figure 10A). Moreover, some CaChi had highest expression levels in all stages of plant growth and development, such as *CaChiI1*, *CaChiIII4*, *CaChiIII5*, *CaChiIV2* and *CaChiVI3*, while some of them had very low or no expression in all the tested tissues, i.e., *CaChiI3*, *CaChiIII1*, *CaChiIII6* and *CaChiVI1*. (Figure 10A). Some genes (*CaChiIV1* and *CaChi13*) are expressed only in particular tissue, mostly in seedling stage. Additionally, to further authenticate the CaChi expression level in various vegetative and reproductive tissues, we cultivated AA3 pepper variety in normal condition and different tissues were collected at different stages. Gene specific primers were used for qRT-PCR analysis (Table S5). As shown in Figure 10B, the expression pattern of CaChi dominantly expressed in seed except *CaChiVI2*, *CaChiVI3*, *CaChiVI4* and *CaChiIII5* expression were maximum in stem/flower, stem and stem, respectively. The lowest expression was recorded in red fruit comparing to other tissues except *CaChiVI1* (529.1), where the particular gene highly correspond to the development of red fruit, flower and leaf. Therefore, it can be assumed that the expression pattern implied by *CaChiVI1* may function importantly in red fruit development. Moreover, *CaChiI2*, *CaChiIII2*, *CaChiIII6*, *CaChiIII7* and *CaChiIV2* did not show highly significant expression to any tested tissues excluding seed but *CaChiIII6* having same results in silico. The integrated investigation of publicly available dataset revealed the ubiquitous expression of these genes and a number of CaChi exhibited a certain degree of tissue specificity. The noticeable differences in both expression level might be related to variation in biological materials, regulation of transcript, interpretation methodology and environmental fluctuation. *2.8. Expression Patterns of CaChi-Genes in Different Tissues*  To further elucidate the expression characteristics of CaChi genes in various vegetative and reproductive tissues (leaf, stem, root, seven developmental phases of placenta and pericarp), we carried out in silico analysis using public transcriptomic database of pepper [39,40]. The different expression patterns of CaChi in pepper exhibited higher variance in distinctive tissues and stages, as demonstrated in heat map where from green to red display the index of expression (Figure 10A). Moreover, some CaChi had highest expression levels in all stages of plant growth and development, such as *CaChiI1*, *CaChiIII4*, *CaChiIII5*, *CaChiIV2* and *CaChiVI3*, while some of them had very low or no expression in all the tested tissues, i.e., *CaChiI3*, *CaChiIII1*, *CaChiIII6* and *CaChiVI1*. (Figure 10A). Some genes (*CaChiIV1* and *CaChi13*) are expressed only in particular tissue, mostly in seedling stage. Additionally, to further authenticate the CaChi expression level in various vegetative and reproductive tissues, we cultivated AA3 pepper variety in normal condition and different tissues were collected at different stages. Gene specific primers were used for qRT-PCR analysis (Table S5). As shown in Figure 10B, the expression pattern of CaChi dominantly expressed in seed except *CaChiVI2*, *CaChiVI3*, *CaChiVI4* and *CaChiIII5* expression were maximum in stem/flower, stem and stem, respectively. The lowest expression was recorded in red fruit comparing to other tissues except *CaChiVI1* (529.1), where the particular gene highly correspond to the development of red fruit, flower and leaf. Therefore, it can be assumed that the expression pattern implied by *CaChiVI1* may function importantly in red fruit development. Moreover, *CaChiI2*, *CaChiIII2*, *CaChiIII6*, *CaChiIII7* and *CaChiIV2* did not show highly significant expression to any tested tissues excluding seed but *CaChiIII6* having same results in silico. The integrated investigation of publicly available dataset revealed the ubiquitous expression of these genes and a number of CaChi exhibited a certain degree of tissue specificity. The noticeable differences in both expression level might be related to variation in biological materials, regulation of transcript, interpretation methodology and environmental

*Int. J. Mol. Sci.* **2018**, *19*, 2216 12 of 26 **Figure 9.** Expression profiles of CaChi in response hormones application. The inducible expression patterns performed by qRT-PCR under Salicylic acid (SA) and methyl-jasmonate (MeJA). Mean

**Figure 10.** Developmental expression profile of chitin-binding protein family gene in pepper. (**A**) The expression pattern retrieved from the database of pepper (CM334), indicating different expression levels of CaChi genes in dissimilar organs. The results were log2 transformed before generating heat maps in leaf, root, stem, 6, 16, 25 days post-anthesis (6DPA, 16DPA, and 25DPA), mature green (MG), breaker (B), 5 and 10 days post-breaker (B5 and B10) of pericarp (PC) and placenta (PL). Three genes (*CaChiI2*, *CaChiIII2* and *CaChiIII7*) were from the Zunla-1 database, so they were not mentioned in the figure. (**B**) The graphs indicate tissue specific expression levels of chitin-binding protein family genes in pepper plant. The samples were collected from different parts root (R), stem (S), leaf (L), flower (F), green fruit (GF), red fruit (RF) and seed (S) analyzed by qRT-PCR. Data are the means of three independent qRT-PCR amplifications. Small letters (a–f) represent significant differences (*p* < 0.05).

(2.7) (Figure 11D).

#### *2.9. Reduced Tolerance of CaChiIV1-Silenced Pepper Plants to NaCl* independent qRT-PCR amplifications. Small letters (a–f) represent significant differences (*p* < 0.05).

To evaluate the role of *CaChiIV1* under NaCl stress, the empty vector (used as control) and *CaChiIV1*-silenced plants were treated with NaCl (300 mM) solution. As shown in Figure 11A, the silencing of *CaChiIV1* significantly compromised resistance to NaCl stress. A greater transcript level of *CaChiIV1* was noted in the control (empty vector) plants than in the *CaChiIV1*-silenced plants in all time points, which is >3 folds. In addition, the transcript levels of other defense-related genes were also studied to see whether the silencing of *CaChiIV1* changes their expression. It was noted that, with the passage of time after NaCl treatment, the expression of *CaDEF1* (defensin) [41] and *CaSAR8.2A* (systemic acquired resistance) [42] were changed, but their rise in the control plants (empty vector) were greater compared to *CaChiIV1*-silenced plants (Figure 11B,C). Additionally, root activity was also studied, and the results revealed a significant decrease in the root activity after NaCl stress. At 24 h post NaCl stress, the root activity of the *CaChiIV1*-silenced plants (1.1) was less than TRV2:00 (2.7) (Figure 11D). *2.9. Reduced Tolerance of CaChiIV1-Silenced Pepper Plants to NaCl* To evaluate the role of *CaChiIV1* under NaCl stress, the empty vector (used as control) and *CaChiIV1*-silenced plants were treated with NaCl (300 mM) solution. As shown in Figure 11A, the silencing of *CaChiIV1* significantly compromised resistance to NaCl stress. A greater transcript level of *CaChiIV1* was noted in the control (empty vector) plants than in the *CaChiIV1*-silenced plants in all time points, which is >3 folds. In addition, the transcript levels of other defense-related genes were also studied to see whether the silencing of *CaChiIV1* changes their expression. It was noted that, with the passage of time after NaCl treatment, the expression of *CaDEF1* (defensin) [41] and *CaSAR8.2A*  (systemic acquired resistance) [42] were changed, but their rise in the control plants (empty vector) were greater compared to *CaChiIV1*-silenced plants (Figure 11B,C). Additionally, root activity was also studied, and the results revealed a significant decrease in the root activity after NaCl stress. At 24 h post NaCl stress, the root activity of the *CaChiIV1*-silenced plants (1.1) was less than TRV2:00

*Int. J. Mol. Sci.* **2018**, *19*, 2216 13 of 26

**Figure 10.** Developmental expression profile of chitin-binding protein family gene in pepper. (**A**) The expression pattern retrieved from the database of pepper (CM334), indicating different expression levels of CaChi genes in dissimilar organs. The results were log2 transformed before generating heat maps in leaf, root, stem, 6, 16, 25 days post-anthesis (6DPA, 16DPA, and 25DPA), mature green (MG), breaker (B), 5 and 10 days post-breaker (B5 and B10) of pericarp (PC) and placenta (PL). Three genes (*CaChiI2*, *CaChiIII2* and *CaChiIII7*) were from the Zunla-1 database, so they were not mentioned in the

green fruit (GF), red fruit (RF) and seed (S) analyzed by qRT-PCR. Data are the means of three

**Figure 11.** The *CaChiIV1*-silenced pepper plants exhibit reduce resistance to NaCl stress: (**A**) The transcript level of *CaChiIV1*; (**B**) transcript level of *CaDEF1*; (**C**) transcript level of *CaSAR82*; and (**D**) root activity of the control and *CaChiIV1*-silenced plants. Values are the means ± SD from three **Figure 11.** The *CaChiIV1*-silenced pepper plants exhibit reduce resistance to NaCl stress: (**A**) The transcript level of *CaChiIV1*; (**B**) transcript level of *CaDEF1*; (**C**) transcript level of *CaSAR82*; and (**D**) root activity of the control and *CaChiIV1*-silenced plants. Values are the means ± SD from three separate experiments. Small letters (a–f) and asterisk (\*significant and \*\*highly significant) denote significant variation (*p* < 0.05).
