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Pullulanase and Starch Synthase III Are Associated with Formation of Vitreous Endosperm in Quality Protein Maize.

Wu H, Clay K, Thompson SS, Hennen-Bierwagen TA, Andrews BJ, Zechmann B, Gibbon BC - PLoS ONE (2015)

Bottom Line: Analysis of recombinant inbred lines derived from a cross of W64Ao2 and K0326Y revealed that pullulanase activity had significant positive correlation with kernel vitreousness.We also found that decreased Starch Synthase III abundance may decrease the pullulanase activity and average glucan chain length given the same Zpu1 genotype.Therefore, Starch Synthase III could indirectly influence the kernel vitreousness by affecting pullulanase activity and coordinating with pullulanase to alter the glucan chain length distribution of amylopectin, resulting in different starch structural properties.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Baylor University, Waco, Texas, 76798, United States of America.

ABSTRACT
The opaque-2 (o2) mutation of maize increases lysine content, but the low seed density and soft texture of this type of mutant are undesirable. Lines with modifiers of the soft kernel phenotype (mo2) called "Quality Protein Maize" (QPM) have high lysine and kernel phenotypes similar to normal maize. Prior research indicated that the formation of vitreous endosperm in QPM might involve changes in starch granule structure. In this study, we focused on analysis of two starch biosynthetic enzymes that may influence kernel vitreousness. Analysis of recombinant inbred lines derived from a cross of W64Ao2 and K0326Y revealed that pullulanase activity had significant positive correlation with kernel vitreousness. We also found that decreased Starch Synthase III abundance may decrease the pullulanase activity and average glucan chain length given the same Zpu1 genotype. Therefore, Starch Synthase III could indirectly influence the kernel vitreousness by affecting pullulanase activity and coordinating with pullulanase to alter the glucan chain length distribution of amylopectin, resulting in different starch structural properties. The glucan chain length distribution had strong positive correlation with the polydispersity index of glucan chains, which was positively associated with the kernel vitreousness based on nonlinear regression analysis. Therefore, we propose that pullulanase and Starch Synthase III are two important factors responsible for the formation of the vitreous phenotype of QPM endosperms.

No MeSH data available.


Related in: MedlinePlus

Sequence analysis of maize Zpu1 and SSIII genes.(A) Multiple alignments of Zpu1 gene sequences between W64A+, W64Ao2 and K0326Y. The highlighted positions showed four single nucleotide polymorphisms in K0326Y. (B) Multiple alignments of translated amino acid sequences of pullulanase. One amino acid difference (highlighted) was found in K0326Y due to the substitution of nucleotide from A to C at position 2864. (C) Restriction analysis of a 3’ end fragment (position 2632–2965) of the Zpu1 gene. The gel bands represent the size of fragments before (uncut) and after digestion (dig) with BslI. (D) Multiple alignments of SSIII gene sequences between W64A+, W64Ao2 and K0326Y. Hash marks on each sequence showed nucleotide polymorphisms. Prior studies identified three regions of the nucleotide sequences: N-terminal region (base 1–2304), homology region (base 2305–3679), and catalytic region (base 3680–5025) [28]. Full nucleotide sequence alignments are provided in S1 Fig. (E) Multiple alignments of translated amino acid sequences of SSIII, and hash marks on each sequence showed amino acid changes. Sequence annotation showed three domains of the amino acid sequences: N-terminal domain (amino acid 1–768) and homology domain (amino acid 769–1226), and catalytic domain (amino acid 1227–1674). Full protein sequence alignments are provided in S2 Fig.
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pone.0130856.g001: Sequence analysis of maize Zpu1 and SSIII genes.(A) Multiple alignments of Zpu1 gene sequences between W64A+, W64Ao2 and K0326Y. The highlighted positions showed four single nucleotide polymorphisms in K0326Y. (B) Multiple alignments of translated amino acid sequences of pullulanase. One amino acid difference (highlighted) was found in K0326Y due to the substitution of nucleotide from A to C at position 2864. (C) Restriction analysis of a 3’ end fragment (position 2632–2965) of the Zpu1 gene. The gel bands represent the size of fragments before (uncut) and after digestion (dig) with BslI. (D) Multiple alignments of SSIII gene sequences between W64A+, W64Ao2 and K0326Y. Hash marks on each sequence showed nucleotide polymorphisms. Prior studies identified three regions of the nucleotide sequences: N-terminal region (base 1–2304), homology region (base 2305–3679), and catalytic region (base 3680–5025) [28]. Full nucleotide sequence alignments are provided in S1 Fig. (E) Multiple alignments of translated amino acid sequences of SSIII, and hash marks on each sequence showed amino acid changes. Sequence annotation showed three domains of the amino acid sequences: N-terminal domain (amino acid 1–768) and homology domain (amino acid 769–1226), and catalytic domain (amino acid 1227–1674). Full protein sequence alignments are provided in S2 Fig.

Mentions: The full length mRNA of the Zpu1 gene has 3,261 nt (acc. AF080567,) [26]. Full length cDNA contigs for Zpu1 from W64A+, W64Ao2 and K0326Y were assembled from 8 overlapping RT-PCR fragments. Multiple sequence alignment of full-length Zpu1 cDNA sequences from W64A+, W64Ao2 and K0326Y was performed using Geneious 5.6.4. The results showed that W64A+ (acc. AF080567) and W64Ao2 (acc. KP872821; W64Ao2-derived Zpu1 allele) had identical Zpu1 alleles, while the Zpu1 allele in K0326Y (acc. KP872822; QPM-derived Zpu1 allele) had 4 single nucleotide polymorphisms (SNPs; Fig 1A). Three of the SNPs were silent, whereas the A→C SNP at position 2864 introduced a change in amino acid sequence from threonine to proline (Fig 1B). This SNP also created a new BslI restriction endonuclease site [27]. A DNA fragment located at the 3’ end of Zpu1 gene (nucleotides 2632–2965) was amplified, purified and digested by BslI to confirm the presence of the SNP. The size did not change for W64A+ and W64Ao2, while K0326Y generated two fragments 240 bp and 94 bp, respectively, indicating the presence of the QPM SNP (Fig 1C).


Pullulanase and Starch Synthase III Are Associated with Formation of Vitreous Endosperm in Quality Protein Maize.

Wu H, Clay K, Thompson SS, Hennen-Bierwagen TA, Andrews BJ, Zechmann B, Gibbon BC - PLoS ONE (2015)

Sequence analysis of maize Zpu1 and SSIII genes.(A) Multiple alignments of Zpu1 gene sequences between W64A+, W64Ao2 and K0326Y. The highlighted positions showed four single nucleotide polymorphisms in K0326Y. (B) Multiple alignments of translated amino acid sequences of pullulanase. One amino acid difference (highlighted) was found in K0326Y due to the substitution of nucleotide from A to C at position 2864. (C) Restriction analysis of a 3’ end fragment (position 2632–2965) of the Zpu1 gene. The gel bands represent the size of fragments before (uncut) and after digestion (dig) with BslI. (D) Multiple alignments of SSIII gene sequences between W64A+, W64Ao2 and K0326Y. Hash marks on each sequence showed nucleotide polymorphisms. Prior studies identified three regions of the nucleotide sequences: N-terminal region (base 1–2304), homology region (base 2305–3679), and catalytic region (base 3680–5025) [28]. Full nucleotide sequence alignments are provided in S1 Fig. (E) Multiple alignments of translated amino acid sequences of SSIII, and hash marks on each sequence showed amino acid changes. Sequence annotation showed three domains of the amino acid sequences: N-terminal domain (amino acid 1–768) and homology domain (amino acid 769–1226), and catalytic domain (amino acid 1227–1674). Full protein sequence alignments are provided in S2 Fig.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4482715&req=5

pone.0130856.g001: Sequence analysis of maize Zpu1 and SSIII genes.(A) Multiple alignments of Zpu1 gene sequences between W64A+, W64Ao2 and K0326Y. The highlighted positions showed four single nucleotide polymorphisms in K0326Y. (B) Multiple alignments of translated amino acid sequences of pullulanase. One amino acid difference (highlighted) was found in K0326Y due to the substitution of nucleotide from A to C at position 2864. (C) Restriction analysis of a 3’ end fragment (position 2632–2965) of the Zpu1 gene. The gel bands represent the size of fragments before (uncut) and after digestion (dig) with BslI. (D) Multiple alignments of SSIII gene sequences between W64A+, W64Ao2 and K0326Y. Hash marks on each sequence showed nucleotide polymorphisms. Prior studies identified three regions of the nucleotide sequences: N-terminal region (base 1–2304), homology region (base 2305–3679), and catalytic region (base 3680–5025) [28]. Full nucleotide sequence alignments are provided in S1 Fig. (E) Multiple alignments of translated amino acid sequences of SSIII, and hash marks on each sequence showed amino acid changes. Sequence annotation showed three domains of the amino acid sequences: N-terminal domain (amino acid 1–768) and homology domain (amino acid 769–1226), and catalytic domain (amino acid 1227–1674). Full protein sequence alignments are provided in S2 Fig.
Mentions: The full length mRNA of the Zpu1 gene has 3,261 nt (acc. AF080567,) [26]. Full length cDNA contigs for Zpu1 from W64A+, W64Ao2 and K0326Y were assembled from 8 overlapping RT-PCR fragments. Multiple sequence alignment of full-length Zpu1 cDNA sequences from W64A+, W64Ao2 and K0326Y was performed using Geneious 5.6.4. The results showed that W64A+ (acc. AF080567) and W64Ao2 (acc. KP872821; W64Ao2-derived Zpu1 allele) had identical Zpu1 alleles, while the Zpu1 allele in K0326Y (acc. KP872822; QPM-derived Zpu1 allele) had 4 single nucleotide polymorphisms (SNPs; Fig 1A). Three of the SNPs were silent, whereas the A→C SNP at position 2864 introduced a change in amino acid sequence from threonine to proline (Fig 1B). This SNP also created a new BslI restriction endonuclease site [27]. A DNA fragment located at the 3’ end of Zpu1 gene (nucleotides 2632–2965) was amplified, purified and digested by BslI to confirm the presence of the SNP. The size did not change for W64A+ and W64Ao2, while K0326Y generated two fragments 240 bp and 94 bp, respectively, indicating the presence of the QPM SNP (Fig 1C).

Bottom Line: Analysis of recombinant inbred lines derived from a cross of W64Ao2 and K0326Y revealed that pullulanase activity had significant positive correlation with kernel vitreousness.We also found that decreased Starch Synthase III abundance may decrease the pullulanase activity and average glucan chain length given the same Zpu1 genotype.Therefore, Starch Synthase III could indirectly influence the kernel vitreousness by affecting pullulanase activity and coordinating with pullulanase to alter the glucan chain length distribution of amylopectin, resulting in different starch structural properties.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Baylor University, Waco, Texas, 76798, United States of America.

ABSTRACT
The opaque-2 (o2) mutation of maize increases lysine content, but the low seed density and soft texture of this type of mutant are undesirable. Lines with modifiers of the soft kernel phenotype (mo2) called "Quality Protein Maize" (QPM) have high lysine and kernel phenotypes similar to normal maize. Prior research indicated that the formation of vitreous endosperm in QPM might involve changes in starch granule structure. In this study, we focused on analysis of two starch biosynthetic enzymes that may influence kernel vitreousness. Analysis of recombinant inbred lines derived from a cross of W64Ao2 and K0326Y revealed that pullulanase activity had significant positive correlation with kernel vitreousness. We also found that decreased Starch Synthase III abundance may decrease the pullulanase activity and average glucan chain length given the same Zpu1 genotype. Therefore, Starch Synthase III could indirectly influence the kernel vitreousness by affecting pullulanase activity and coordinating with pullulanase to alter the glucan chain length distribution of amylopectin, resulting in different starch structural properties. The glucan chain length distribution had strong positive correlation with the polydispersity index of glucan chains, which was positively associated with the kernel vitreousness based on nonlinear regression analysis. Therefore, we propose that pullulanase and Starch Synthase III are two important factors responsible for the formation of the vitreous phenotype of QPM endosperms.

No MeSH data available.


Related in: MedlinePlus