A novel α‐amylase gene is transiently upregulated during low temperature exposure in apple fruit

Abstract

An a-amylase gene product was isolated from apple fruit by reverse-transcriptase PCR using redundant primers, followed by 5 H and 3 H RACE. The gene is a member of a small gene family. It encodes a putative 46.9 kDa protein that is most similar to an a-amylase gene from potato (GenBank accession M79328). In apple fruit this new gene was expressed at low levels, as detected by reverse-transcriptase PCR, in a number of plant tissues and during fruit development. Highest levels of mRNA for this transcript were observed 3 to 9 days after placing apple fruit at 0.5 8C. Phylogenetic analysis of amino acid sequence places the potato and apple proteins as a distinct and separate new subgroup within the plant a-amylases, which appears to have diverged prior to the split between monocotyledonous and dicotyledonous plants. These two divergent a-amylases lack the standard signal peptide structures found in all other plant a-amylases, and have sequence differences within the B-domain and C-domain. However, comparisons with structures of known starch hydrolases suggest that these differences are unlikely to affect the enzymatic a-1,4-amylase function of the protein. This is the first report of upregulation of a dicotyledonous a-amylase in response to low temperature, and confirms the presence of a new family of a-amylases in plants.Keywords: apple; a-amylase; low temperature; phylogenetic analysis; protein model. a-Amylase (EC 3.2.1.1) is involved in the hydrolysis of a-1,4-glucan bonds and functions in plants as the major starch hydrolase. The most commonly studied a-amylases in plants are those from the monocotyledonous cereals rice, wheat and barley. Only one protein structure for a plant a-amylase has been determined by X-ray crystallography, that of the high pI isozyme from Hordeum vulgare grains [1]. It consists of the catalytic A-domain in the form of a (ba) 8 barrel, a B-domain that protrudes from the barrel and a five-stranded b-sheet C-domain. The interface between the A and B-domains is stabilized by calcium ions. The general structure of the catalytic barrel is conserved in starch hydrolases from many organisms, as are a number of invariant residues at the active site [2]. In barley a-amylases the B-domain is involved in determining substrate specificity [3].Most a-amylase genes have been isolated from germinating cereal grains, which have extensive starch hydrolysis carried out by enzymes secreted into the endosperm. Individual a-amylase genes vary in their spatial and temporal pattern of transcription, partly in response to hormonal and metabolic signals during germination. Increased transcription of some, but not all, of these genes has also been observed in other tissues, such as leaves under water stress [4], but at levels significantly lower than those during germination. In comparison with the extensive range of a-amylases from monocotyledons, only five have been reported from dicotyledons, with limited expression data. Two are from germinating seeds of Vigna mungo (mung bean) [5,6] and Phaseolus vulgaris [...

Isolation and characterization of an α-amylase gene in cassava (Manihot esculenta)

“…3). Our phylogenetic tree is very similar to the one reported by Wegrzyn et al [39], although potato Amy23 and apple Amy8 do not form an outgroup in this tree. This is because a greater number of dicot a-amylase sequences from A. thaliana, kiwifruit (Actinidia chinensis), apple (Amy8, Amy9 and Amy10) and morning glory were taken into account in this analysis.…”

Spatiotemporal Profiling of Starch Biosynthesis and Degradation in the Developing Barley Grain

“…The latter shares only limited homology with the three known barley AMY sequences but is highly homologous to a putative rice AMY (GenBank accession no. Os04g0403300) and to a large number of dicot sequences, including those from Plantago major (71.2% identity; Pommerrenig et al, 2006), Malus domestica (70.0% identity; Wegrzyn et al, 2000), and Arabidopsis (68.0% identity; GenBank accession no. At1g76130).…”

Teores de carboidratos em estacas lenhosas de mirtileiro

“…Esse fato ocorre no período de dormência quando os tecidos meristemáticos das gemas apresentam baixa capacidade mobilizadora de açúcares solúveis, acompanhado de aumento da hidrólise do amido, sob baixas temperaturas, pela ação da enzima α-amilase (WEGRZYN et al, 2000), e síntese de sacarose através da sacarose fosfato sintase -SPS -(SCHRADER; SAUTER, 2002), como forma de proteção desses órgãos ao frio.…”

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