# Cloning and Functional Identification of _Sipg49_ in _Solanum Lyopersicum_
## 4 Discussion  
According to the latest classification system, PGs can be divided into seven clades (A-G), and most members of clade E can be detected at a high overall expression in all tissues [45]. In the present work, phylogenetic analysis showed that _SIPG49_ was located in clade E and closed to _ATPG5_ (Figure 1D). Interestingly, the expression of _ATPG5_ was significantly increased when an _Arabidopsis_ endo-PG (_AIPGLR_) was knocked out, indicating that _ATPG5_ has the function of replenishing _AtPGLR_, which may also be an endo-PG [29]. Therefore, in view of the close evolutionary relationship between SIPG49 and ATPG5, we speculated that _SIPG49_ was probably an endo-PG. As mentioned earlier, PG has four conserved domains (I-IV). Generally, genes encoding the proteins of these four domains are defined as PG genes. Notably, domain III is relatively poorly conserved [46]. The results of multiple sequence alignments show that the conservation of domain III is indeed lower than that of domains I, II, and IV (Figure 1D). Although they are different from the typical domain III, the nine PGs from clade E (including SIPG49) are highly conserved in domain III. It has been reported that the positively charged domain IV may participate in an ionic interaction with the carboxyl terminal of the substrate [47]. Here, although R (Arginine) in the domain IV of SIPG49 is replaced by H (Histidine), it is still positively charged. Therefore, we speculated that the structure and function of SIPG49 may not have been changed. A previous study showed that the enzymatic activity of PG increased during the ripening period of the tomato fruit, suggesting that these enzymes may be critical for initiating the ripening process [21]. In our previous research, _SIPG49_ was predicted to have a high expression in red ripening fruits [18]. In this work, qPCR analyses confirmed that _SIPG49_ is extremely highly expressed in fruits at the red ripening stage (Figure 2), demonstrating that _SIPG49_ may be involved in tomato fruit ripening and softening. Pectin is synthesized in the Golgi apparatus and secreted into the cell wall in the state of methyl esterification and acetylation, and can be modified and degraded in the cell wall [26]. PGs are synthesized in the endoplasmic reticulum and secreted into the cell wall after maturation in the trans-Golgi network [48; 49]. In accordance with their function, it is predicted that PGs exhibit extracellular localization. Previous studies have demonstrated that a PG protein in _Brassica campus_, BcMF23, was a secreted protein and exhibited extracellular localization by subcellular localization of onion epidermis transient expression [50]. In transgenic Arabidopsis, _ATPGX3_ and _AIPG45_ were located in the cell wall [51; 52]. The present transient expression result in the onion epidermis confirmed that SIPG49 was a secreted protein and may play a great role in the cell wall or  
Figure 4: SIPG49 displays PG activity in vitro: (**A,B**) signal peptide analysis and schematic protein structure of SIPG49; (**C**) SDS-PAGE analysis; M, protein maker; 1, not induced pMAL-MBP; 2, induced pMAL-MBP; 3, not induced pMAL-MBP-SIPG49; 4, induced pMAL-MBP-SIPG49. White arrowheads show the bands of pMAL-MBP-SIPG49 and pMAL-MBP; (**D**) western blot analysis; 1: purified pMAL-MBP; 2: purified pMAL-MBP-SIPG49; (**E**) purified pMAL-MBP-SIPG49 shows PG activity in vitro. Data are presented as mean \(\pm\) SE with at least three biological replicates. Significant differences are indicated by different letters (\(p\) < 0.05). extracellular matrix (Figure 3), similar to PGs in _A. thaliana_. To date, only nine PG genes have been identified in _A. thaliana_, with five that have been proven to show PG activity in vitro [51; 53; 54; 55]. However, there is no report of in vitro activity of PGs in tomatoes.