Isolation of a cDNA Clone for α-Amylase in Mung Bean Cotyledons

Abstract

A cDNA was isolated that codes for a-amylase in mung bean (Vigna radiata) cotyledons, and the nucleotide sequence was determined. The deduced amino acid sequence (421 amino acid residues) is about 65% homologous with those of barley a-amylases. By comparing the deduced sequence with the sequence of the purified a-amylase, it was inferred that 23 Nterminal amino acids of a nascent polypeptide represent a signal peptide. Northern blot analysis showed that the levels of a-amylase mRNA are in parallel with the activities of a-amylase synthesis in cotyledons. Under the conditions where the solute leakage from cotyledons is accelerated during imbibition, a rapid increase in the amount of the a-amylase mRNA occurs. We postulate that a factor(s) which regulates in an inhibitory manner the a-amylase expression at the transcriptional level may be present in dry cotyledons and be removed by leakage.The mechanism for the development of a-amylase in reserve tissues of cereal seeds has been studied in detail; GA secreted by the embryo to the scutellum or the aleurone layer induces the synthesis of the enzyme by causing the synthesis of new a-amylase mRNA (13). In contrast, in legume seeds, the mechanism for the development of a-amylase is much less clear; hormonal control of a-amylase development such as seen in cereals has not been demonstrated unambiguously. Although much work has been done on the role of the embryonic axis in the development of the enzyme in reserve tissues (cotyledon), the results are inconsistent (6,10,12,19,20).In a previous report (1 1 no dicot amylase cDNA has been available. In the present study we have cloned cDNA for mung bean a-amylase and examined the levels of a-amylase transcript in cotyledons. MATERIALS AND METHODS Plant MaterialsSeeds of mung bean, Vigna radiata (L.) Wilczek, were treated as previously described (11). Briefly, H2S04-treated seeds were soaked in water for 6 h and husked, and the two cotyledons of each seed were separated, the embryonic axis remaining attached to one of them. The AC2 and the DC were placed on wet filter paper and incubated at 28°C in the dark. In some experiments, the two cotyledons of a seed were separated prior to imbibition, the axis being detached. The adaxial side of such cotyledons is not covered with the seed coat. The cotyledons were soaked in water for 6 h or 15 min. In the former case (6-h imbibition), the seed coats covering the abaxial surface of cotyledons were removed after imbibition and the cotyledons were incubated as above (DDC). In the latter case, the cotyledons were desiccated after 15-min imbibition, resoaked in water for 6 h, husked, and incubated as above (RIC). In another set of experiments, detached cotyledons separated from dry seeds were allowed to imbibe water by placing them abaxial side down on wet filter paper for 6 h, and then they were husked, transferred onto wet filter paper, and incubated as above (FPC)

Classification and evolution of alpha-amylase genes in plants.

“…The design of these primers was based on the following two criteria: (i) both primers correspond to highly conserved regions in the a-amylase genes and (ii) the PCR products for the AmyA and AmyB genes could be easily resolved by polyacrylamide gel electrophoresis. The two mung bean a-amylase gene primers were based on the previously published sequence information (30).…”

“…Since zymograms do not resolve the a-amylase isozymes of rice into distinct high and low pI groups (2,19), it has been difficult to relate these isozymes to those observed in barley and wheat. Subsequently, Huang et al (20) used DNA-DNA hybridization to classify 30 rice genomic clones into five hybridization groups. These five groups were eventually consolidated into three subfamilies (Amyl, Amy2, and Amy3) on the basis of protein comparisons to other wheat and barley a-amylase genes (8,(20)(21)(22).…”

Improved Activity and Modulated Action Pattern Obtained by Random Mutagenesis at the Fourth β-α Loop Involved in Substrate Binding to the Catalytic (β/α)8-Barrel Domain of Barley α-Amylase 1

“…respectively (9,34,35,46,56 (6). Furthermore, NE2 of His 201 from the Lys-His-Z motif in PPA forms a hydrogen bond with sugar OH-2 in subsite ϩ1, i.e.…”

“…A conserved region in the ␤-␣ loop extending at the C terminus of the fourth ␤-strand of the (␤/␣) 8 -fold is interpreted to play an important role in the specificity (25, 27, 30 -32). Remarkably, Arg 183 -Gly 184 -Tyr 185 at this loop in AMY1 is distinctive to plant ␣-amylases; others have Lys-His-Z, where Z is hydrophobic (9,25,27,(33)(34)(35). Arg 183 in AMY1 aligns with Lys 182 in AMY2 (9), which in the crystal structure of AMY2-acarbose (71) forms a hydrogen bond to OH-3 of the sugar ring at subsite ϩ2 and in the crystal structure of AMY2-BASI (36) interacts with a glu-…”

Purification, Characterization, and Sequence Analysis of Two α-Amylase Isoforms from Azuki Bean,Vigna angularis, Showing Different Affinity towards β-Cyclodextrin Sepharose

“…7) In leguminous plants, a-amylase is expressed in cotyledons during seed germination, where its mRNA was isolated. [8][9][10] The a-amylase is localized in lytic vacuoles in cotyledon cells, and starch granules are transported into the vacuoles via an autophagic process, and hydrolyzed there. 11) An Arabidopsis mutant suggested that one of the a-amylases in the plant is responsible for degradation of transitory starch in chloroplasts.…”