The calculated molecular weights for the transit peptide and mature protein of rye isoamylase are 5.21 kD and 83.56 kD, respectively. The predicted pI for the mature isoamylase is 5.46. The aa sequences of mature isoamylases exhibited more than 83% homology LDK378 solubility dmso among rye and other plant genomes, but especially more than 95% homology between rye and Ae. tauschii, wheat and barley. However, sequence homologies for the transit peptides of isoamylases between rye and rice or maize are 31.75% or 27.59%, respectively, significantly less than similar comparisons for the mature proteins (83.31% or 87.18%, respectively)

( Table 3). Our results indicate that the structural conservation of the transit peptides for this enzyme is generally lower than that of the mature proteins. Since the transit peptides are the N-terminal aa presequences that direct proteins to an organelle (e.g., chloroplast, mitochondria) and are required for their

transport across membranes from their synthesis sites in the cytoplasm [29], significant diversities in transit peptides of isoamylase between rye and rice or maize may be related to their different cellular structures Sotrastaurin cell line and metabolic functions, although the mature isoamylases share similar catalytic domains and elements. We used quantitative real-time PCR to analyze the expression of the rye isoamylase gene in various tissues else and at different seed developmental stages. Our results showed that the isoamylase gene

is expressed in all rye tissues tested in this study, with seeds having significantly higher levels of isoamylase transcript than leaves, stems and roots (Fig. 3-A). A recent study showed that the ISA1 transcript level is relatively abundant in maize tissues where starch is synthesized [32]. As the leaf and other green tissues are temporary storage places for starch accumulation during photosynthesis, the expression of the isoamylase gene in rye leaves and stems demonstrated that amylase may have an important role for either starch synthesis or starch degradation. Isoamylase is termed as the debranching enzyme, essential for formation of crystalline amylopectin [6]. We analyzed the expression profiles of the rye isoamylase gene during endosperm development and found that its expression in rye endosperm reached a maximum level at the mid-development stage (15 DPA) and then dropped through 24 and 33 DPA (Fig. 3-B). Consistent with previous reports on wheat and maize [23] and [32], our results confirmed that the isoforms of isoamylase-type DBE genes are maximally expressed during endosperm development and then gradually decline during grain maturation. Studies on barley mutants and transgenic rice suggested that isoamylases play a crucial role in synthesis of phytoglycogen and starch granule structure and initiation [14] and [19].