A single limit dextrinase gene is expressed both in the developing endosperm and in germinated grains of barley

Abstract

The single gene encoding limit dextrinase (pullulan 6-glucanohydrolase; EC 3.2.1.41) in barley (Hordeum vulgare) has 26 introns that range in size from 93 to 822 base pairs. The mature polypeptide encoded by the gene has 884 amino acid residues and a calculated molecular mass of 97,417 D. Limit dextrinase mRNA is abundant in gibberellic acid-treated aleurone layers and in germinated grain. Gibberellic acid response elements were found in the promoter region of the gene. These observations suggest that the enzyme participates in starch hydrolysis during endosperm mobilization in germinated grain. The mRNA encoding the enzyme is present at lower levels in the developing endosperm of immature grain, a location consistent with a role for limit dextrinase in starch synthesis. Enzyme activity was also detected in developing grain. The limit dextrinase has a presequence typical of transit peptides that target nascent polypeptides to amyloplasts, but this would not be expected to direct secretion of the mature enzyme from aleurone cells in germinated grain. It remains to be discovered how the enzyme is released from the aleurone and whether another enzyme, possibly of the isoamylase group, might be equally important for starch hydrolysis in germinated grain.

Figure 1

a, Partial restriction map of the 3429-bp cDNA encoding barley limit dextrinase. b, Overlapping cDNAs and PCR products used to obtain the complete nucleotide sequence of the cDNA for barley limit dextrinase. The 2.6-kb 3′ cDNA was screened from a library of GA₃-treated aleurone layers. The 316-bp “central” sequence was obtained as a PCR product amplified from cDNA inserts excised from the same aleurone cDNA library. The 507-bp 5′ fragment was prepared by 5′ RACE PCR from aleurone total RNA. The arrows at the 3′ end indicate multiple polyadenylation sites.

Figure 2

Nucleotide sequence of the overlapping cDNAs for barley limit dextrinase and the deduced amino acid sequence for the enzyme. The NH₂-terminal Ala of the mature polypeptide is indicated by the vertical arrow, and the amino acid sequence obtained directly from the purified enzyme is underlined and in bold. Potential N-glycosylation sites are underlined and amino acid residues that are likely to be involved in catalysis are in bold. The TGA stop codon is also shown in bold and the four large arrows near the 3′ end show the various positions of polyadenylic acid tails in different cDNAs isolated. The putative polyadenylation signal AATAAA is underlined. The cDNA accession number is AF122049.

Figure 2

Nucleotide sequence of the overlapping cDNAs for barley limit dextrinase and the deduced amino acid sequence for the enzyme. The NH₂-terminal Ala of the mature polypeptide is indicated by the vertical arrow, and the amino acid sequence obtained directly from the purified enzyme is underlined and in bold. Potential N-glycosylation sites are underlined and amino acid residues that are likely to be involved in catalysis are in bold. The TGA stop codon is also shown in bold and the four large arrows near the 3′ end show the various positions of polyadenylic acid tails in different cDNAs isolated. The putative polyadenylation signal AATAAA is underlined. The cDNA accession number is AF122049.

Figure 3

Structure of an 11.5-kb genomic DNA fragment carrying the barley limit dextrinase gene, showing the 27 exons (black), the 26 introns (white), and the promoter region (shaded). Positions of the GA₃ response elements (GARE), the putative TATA box of the promoter, the NH₂ terminus of the mature protein, and the stop codon are indicated.

Figure 4

Promoter region of the barley limit dextrinase gene. a, Nucleotide sequence of the gene upstream from the translation start ATG codon. The putative transcription start point is indicated by the arrowhead and is designated nucleotide +1. Underlined sequences starting from the 5′ end of the sequence include the CCTTT pyrimidine box, the TAAAACAAA box and the TATCCAA box of the putative GA₃ response element, and the CATT box and the proposed TATA box of the gene. b, Primer extension from total RNA of GA₃-treated aleurone layers using a primer specific for the 5′ region of the cDNA. The top two lanes show the major primer extension (PE) termination products (arrows), whereas the lower four lanes represent the nucleotide sequence of the gene, primed with the same oligonucleotide used for the primer extension.

Figure 5

Northern blots of RNA preparations from developing barley grain and GA₃-treated aleurone layers using limit dextrinase cDNA as a probe. a, Lanes 1 to 4, RNA from grain during the period 19 to 40 DPA (grain weight 27–70 mg); lanes 5 and 6, RNA from aleurone layers treated for 48 h with (+GA) or without (−GA) 2 μm GA₃. b, Time course of GA₃ induction of limit dextrinase mRNA transcripts in isolated barley aleurone layers.

Figure 6

RT-PCR analysis of limit dextrinase mRNA transcripts in the developing barley grain 6 to 36 DPA. Lane M, DNA molecular mass markers; lane C, control PCR reactions; lanes 1 to 65, weight of the grain (in milligrams) at the time of RNA extraction. Below are shown the corresponding times of RNA extraction (6–36 DPA). a, RT-PCR products amplified with limit dextrinase (LD) primers; b, Southern blot of the RT-PCR products shown after probing with a limit dextrinase cDNA; c, RT-PCR products amplified with primers for the constitutively expressed GAPDH after 35 cycles; d, RT-PCR products of GAPDH primers after 28 cycles; e, limit dextrinase (LD) primers and 35 PCR cycles of RNA preparations extracted from the pericarp (P) and endosperm (E) of developing grain, together with products amplified in 28 cycles using the GAPDH primers.

Figure 7

IEF of extracts of a barley developing grain (A) and a germinated grain (B). The left panels show proteins stained with Coomassie brilliant blue and the right panels show limit dextrinase activity revealed by clearing of a Red Pullulan overlay gel. Lanes S, pI protein standards; lanes M, mature grain and other extracts from developing endosperm 8, 10, and 16 DPA. The number of days after the initiation of germination are indicated in B.

Figure 8

Amino acid sequence alignments of the catalytic domains of debranching enzymes from barley (Fig. 2), rice (Nakamura et al., 1996), spinach (A. Renz, R. Schmid, J. Kossmann, and E. Beck, unpublished data), and K. aerogenes (Katsuragi et al., 1987), using the PileUp and PrettyBox programs (Devereux et al., 1984). Shaded and hatched boxes indicate identical and homologous residues, respectively.

Figure 9

Unrooted, radial phylogenetic tree of selected starch-debranching enzymes as determined using the PileUp, EprotDist, and EFitch programs of the University of Wisconsin package (version 8; Devereux et al., 1984). The two major branches distinguish the pullulanase type (upper branch) from the isoamylase type (lower branch). The accession numbers of the sequences are shown.