4.1 PpINHs are cold-responsive regulators that have evolved with flowering plants
Plant invertase inhibiters are small proteins that have been identified in a variety of plant species, such as tobacco (Greiner et al. 1998; Weil, Krausgrill et al. 1994), Arabidopsis (Link et al.2004), maize (Bate et al. 2004), tomato (Reca et al., 2008), potato (Brummell et al., 2011), and sugarcane (Shivalingamurthy et al., 2018). Wan et al. 2018, have reported that INHs, have evolved with vascular plants; our BLASTP results support this, as we did not identify any INHs from lower plants such as algae and moss (Figure 1a, b ), and inAmborella trichopoda, the common ancestor of living angiosperms (Albert et al., 2013), no INH gene was predicted, which supports the thesis that INHs evolved in flowering plants. Although the number ofINH genes varies among species, INH gene duplication appears to have followed species evolution, indicating their importance in modern vascular plants. Amino acid sequence alignments of INHs from different species showed that all contained four highly conserved cysteine (Cys) residues, a hallmark of plant INHs (Rausch, & Greiner, 2004). The nucleic acid sequences however, vary among species, even within a species (Figure 1c) .
CBF is a type of AP2/ERF transcription factor; it binds to CRT/DRE cis- elements in the promoter region of target genes regulating their expression. In wild-type Arabidopsis , 302 genes are induced by low temperature, in Arabidopsis constitutively expressing CBF, only 85 genes are induced by low temperature. Moreover, among genes downstream from CBF, 8 were inhibited when CBF was overexpressed and these 8 genes are down-regulated by low temperature (Vogel et al., 2005). These results suggest CBF plays a key role in plant cold response. We found numerous CRT/DRE cis-elements in 5 PpINHpromoters (Figure 2 ), indicating these PpINHs are targets of CBF. Expression of PpINH1 was significantly reduced in peaches during cold storage (Figure 6f ) indicatingPpINH1 is sensitive to cold stress. These data suggest that low-temperature sensitive INHs may be regulated by CBF proteins.