Molecular Cloning and Expression Analysis of Triticale Phytocystatins During Development and Germination of Seeds

Abstract

Three triticale cDNAs encoding inhibitors of cysteine endopeptidases, belonging to phytocystatins, have been identified and designated as TrcC-1, TrcC-4 and TrcC-5. Full-length cDNAs of TrcC-1 (617 bp) and TrcC-4 (940 bp), as well as a fragment of TrcC-5 cDNA (369 bp), were obtained. A high-level identity of the deduced amino acid sequence of TrcCs with other known phytocystatins, especially with wheat and barley, has been observed. Moreover, the presence of conserved domain, containing the G and W residues, the sequence of QxVxG and the sequence of LARFAV, characteristic for plant cysteine endopeptidase inhibitors, has been noted. The profiles of TrcC-1 and TrcC-5 mRNA levels in the developing seeds of two triticale cultivars that differ in their resistance to preharvest sprouting (Zorro and Disco) were similar. However, the expression of TrcC-4 was, higher in the developing seeds, and in the scutellum of germinating seeds of a cultivar more resistant to preharvest sprouting (Zorro) than in the less resistant (Disco). Additionally, the expression of TrcC-4 remained longer in developing seeds of Zorro as compared to Disco. The performed studies suggest that TrcC-4 might have an influence on the higher resistance of Zorro cultivar to preharvest sprouting.

The participation of phytocystatin TrcC-4 in the activity regulation of EP8, the main prolamin degrading cysteine endopeptidase in triticale seeds

“…3A). These results correspond to the high mRNA level of TrcC-4 observed during the first 2 weeks of development, just before the accumulation of storage proteins (Szewińska et al 2012). The high mRNA level of wheat phytocystatin, WC4 (84 % amino acid sequence identity with TrcC-4), was also observed during the first 2 weeks after pollination (Kuroda et al 2001).…”

“…As we previously demonstrated, TrcC-4 belongs to group A of the PCs from Poaceae family (Szewińska et al 2012), the inhibitors that may regulate the activity of endogenous CEPs (Arai et al 2002;Corre-Menguy et al 2002). Western blot analysis, using anti-TrcC-4 antibodies, demonstrated the relatively high level of TrcC-4 protein in developing grains of triticale in the initial stage of the process, decreasing in later stages (Fig.…”

“…Our previous studies indicated that mRNA levels of TrcC-4 were higher and lasted longer in the triticale cultivar more resistant to pre-harvest sprouting (Zorro) than in a less resistant cultivar (Disco) (Szewińska et al 2012). In this paper we analysed the protein level of TrcC-4 during development and germination of mentioned cultivars.…”

Identification and expression analysis of a novel phytocystatin in developing and germinating seeds of triticale (×Triticosecale Wittm.)

“…There are 13 known phytocystatins in barley [11], 12 in rice [12,13], 10 in maize [14] and 6 in wheat [15,16], 5 in triticale [2]. The new PhyCys described in this paper, TrcC-7, is characterized by conserved regions that are present in most cystatin superfamily inhibitors and most likely has signal peptide (Fig.…”

“…), and one (TrcC-4) has been shown to have inhibitory activity against endogenous cysteine proteinase EP8, what may be related to pre-harvest sprouting tolerance [2][3][4]. Therefore, we examined another triticale phytocystatin.…”