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Identification and characterization of the maize arogenate dehydrogenase gene family.

Holding DR, Meeley RB, Hazebroek J, Selinger D, Gruis F, Jung R, Larkins BA - J. Exp. Bot. (2010)

Bottom Line: In plants, the amino acids tyrosine and phenylalanine are synthesized from arogenate by arogenate dehydrogenase and arogenate dehydratase, respectively, with the relative flux to each being tightly controlled.A Mutator insertion at an equivalent position in AroDH-3, the most closely related family member to AroDH-1, is also associated with opaque endosperm and stunted vegetative growth phenotypes.Overlapping but differential expression patterns as well as subtle mutant effects on the accumulation of tyrosine and phenylalanine in endosperm, embryo, and leaf tissues suggest that the functional redundancy of this gene family provides metabolic plasticity for the synthesis of these important amino acids. mto140/arodh-1 seeds shows a general reduction in zein storage protein accumulation and an elevated lysine phenotype typical of other opaque endosperm mutants, but it is distinct because it does not result from quantitative or qualitative defects in the accumulation of specific zeins but rather from a disruption in amino acid biosynthesis.

View Article: PubMed Central - PubMed

Affiliation: Center for Plant Science Innovation, University of Nebraska, 1901 Vine St., Lincoln, NE 68588, USA. dholding2@unl.edu

ABSTRACT
In plants, the amino acids tyrosine and phenylalanine are synthesized from arogenate by arogenate dehydrogenase and arogenate dehydratase, respectively, with the relative flux to each being tightly controlled. Here the characterization of a maize opaque endosperm mutant (mto140), which also shows retarded vegetative growth, is described The opaque phenotype co-segregates with a Mutator transposon insertion in an arogenate dehydrogenase gene (zmAroDH-1) and this led to the characterization of the four-member family of maize arogenate dehydrogenase genes (zmAroDH-1-zmAroDH-4) which share highly similar sequences. A Mutator insertion at an equivalent position in AroDH-3, the most closely related family member to AroDH-1, is also associated with opaque endosperm and stunted vegetative growth phenotypes. Overlapping but differential expression patterns as well as subtle mutant effects on the accumulation of tyrosine and phenylalanine in endosperm, embryo, and leaf tissues suggest that the functional redundancy of this gene family provides metabolic plasticity for the synthesis of these important amino acids. mto140/arodh-1 seeds shows a general reduction in zein storage protein accumulation and an elevated lysine phenotype typical of other opaque endosperm mutants, but it is distinct because it does not result from quantitative or qualitative defects in the accumulation of specific zeins but rather from a disruption in amino acid biosynthesis.

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Expression of AroDH family genes in maize tissues. (A) RT-PCR analysis of AroDH-1–AroDH-4 in arodh-1 mutant and wild-type tissues. (B) Real-time qRT-PCR analysis of AroDH-2 and AroDH-3 showing their expression relative to AroDH-1 expression in endosperm, embryo, and leaf tissues (shown as 1 relative expression unit). (C) Real-time qRT-PCR analysis of embryo and leaf expression relative to endosperm expression for AroDH-1, AroDH-2, and AroDH-3 (each gene relative to its own endosperm expression level; 1 relative expression unit). The key (black=endosperm, grey=embryo, white=leaf) for B also applies to C.
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fig5: Expression of AroDH family genes in maize tissues. (A) RT-PCR analysis of AroDH-1–AroDH-4 in arodh-1 mutant and wild-type tissues. (B) Real-time qRT-PCR analysis of AroDH-2 and AroDH-3 showing their expression relative to AroDH-1 expression in endosperm, embryo, and leaf tissues (shown as 1 relative expression unit). (C) Real-time qRT-PCR analysis of embryo and leaf expression relative to endosperm expression for AroDH-1, AroDH-2, and AroDH-3 (each gene relative to its own endosperm expression level; 1 relative expression unit). The key (black=endosperm, grey=embryo, white=leaf) for B also applies to C.

Mentions: Given that the Mu insertion in arodh-1 occurs in a region encoding the C-terminal domain of the protein and that the mutant phenotype is non-lethal, it was of interest to determine whether a transcript could be detected. A truncated protein could retain some enzymatic function, since the predicted active site (Legrand et al., 2006) is intact, and only the predicted dimerization domain (Legrand et al., 2006) is disrupted. Also, since the maize arogenate dehydrogenase family is predicted to have redundant function, it was of interest to determine whether a compensatory increase in the expression of other gene family members could be detected in the arodh1 mutant. Using RT-PCR, expression of AroDH-1, AroDH-2, AroDH-3 and AroDH-4 was detected in endosperm, embryo, and leaf tissues of both arodh-1 and wild-type plants (Fig. 5A). Primers upstream of the Mu insertion where the genes are very GC rich were designed, necessitating use of GC polymerase (Clontech) and high annealing temperatures for the PCRs. Since ethidium bromide staining of stationary phase RT-PCR products is not suitable for detecting quantitative differences in transcript abundance, it was not possible to draw conclusions on the relative expression of AroDH family members from this analysis. Therefore, real-time RT-PCR was used to quantify expression of the AroDH genes in these tissues. In this case, primers were designed to the divergent 3′-untranslated regions so annealing temperatures compatible with the real-time PCR were possible. Despite several different designs, AroDH-4 primers were not suitable for real-time PCR, since they gave multiple products. The expression of each gene was measured in the arodh-1 background and it was expressed relative to the wild type for each tissue type. This analysis showed that, as expected, no transcript is produced downstream of the Mu insertion site in arodh-1 and, furthermore, that AroDH-2 and AroDH-3 expression is not increased in arodh-1 mutant tissues (not shown). Wild-type endosperm, embryo, and leaf RNAs were then used to address the relative expression levels of AroDH-2 and AroDH-3, in comparison with AroDH-1, and to determine the tissue specificity of each gene family member. In Fig. 5B the expression of AroDH-2 and AroDH-3 in each tissue relative to the AroDH-1 expression level for each tissue type is shown. In endosperm, AroDH-2 is expressed at 18% the level of AroDH-1, and AroDH-3 at 60% of that level. AroDH-2 has the highest relative expression of the three genes in the leaf tissue, and AroDH-3 has the highest relative expression of the three genes in the embryo tissue. In Fig. 5C the expression of each gene in embryo and leaf tissue relative to its own endosperm expression level is shown to demonstrate the tissue specificity of each gene. These data show that AroDH-1 is predominantly an endosperm-expressed gene, whereas AroDH-2 shows little tissue specificity, although it is most highly expressed in the leaf. AroDH-3 expression is biased towards the seed and shows a greater relative embryo expression than does AroDH-1.


Identification and characterization of the maize arogenate dehydrogenase gene family.

Holding DR, Meeley RB, Hazebroek J, Selinger D, Gruis F, Jung R, Larkins BA - J. Exp. Bot. (2010)

Expression of AroDH family genes in maize tissues. (A) RT-PCR analysis of AroDH-1–AroDH-4 in arodh-1 mutant and wild-type tissues. (B) Real-time qRT-PCR analysis of AroDH-2 and AroDH-3 showing their expression relative to AroDH-1 expression in endosperm, embryo, and leaf tissues (shown as 1 relative expression unit). (C) Real-time qRT-PCR analysis of embryo and leaf expression relative to endosperm expression for AroDH-1, AroDH-2, and AroDH-3 (each gene relative to its own endosperm expression level; 1 relative expression unit). The key (black=endosperm, grey=embryo, white=leaf) for B also applies to C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2921203&req=5

fig5: Expression of AroDH family genes in maize tissues. (A) RT-PCR analysis of AroDH-1–AroDH-4 in arodh-1 mutant and wild-type tissues. (B) Real-time qRT-PCR analysis of AroDH-2 and AroDH-3 showing their expression relative to AroDH-1 expression in endosperm, embryo, and leaf tissues (shown as 1 relative expression unit). (C) Real-time qRT-PCR analysis of embryo and leaf expression relative to endosperm expression for AroDH-1, AroDH-2, and AroDH-3 (each gene relative to its own endosperm expression level; 1 relative expression unit). The key (black=endosperm, grey=embryo, white=leaf) for B also applies to C.
Mentions: Given that the Mu insertion in arodh-1 occurs in a region encoding the C-terminal domain of the protein and that the mutant phenotype is non-lethal, it was of interest to determine whether a transcript could be detected. A truncated protein could retain some enzymatic function, since the predicted active site (Legrand et al., 2006) is intact, and only the predicted dimerization domain (Legrand et al., 2006) is disrupted. Also, since the maize arogenate dehydrogenase family is predicted to have redundant function, it was of interest to determine whether a compensatory increase in the expression of other gene family members could be detected in the arodh1 mutant. Using RT-PCR, expression of AroDH-1, AroDH-2, AroDH-3 and AroDH-4 was detected in endosperm, embryo, and leaf tissues of both arodh-1 and wild-type plants (Fig. 5A). Primers upstream of the Mu insertion where the genes are very GC rich were designed, necessitating use of GC polymerase (Clontech) and high annealing temperatures for the PCRs. Since ethidium bromide staining of stationary phase RT-PCR products is not suitable for detecting quantitative differences in transcript abundance, it was not possible to draw conclusions on the relative expression of AroDH family members from this analysis. Therefore, real-time RT-PCR was used to quantify expression of the AroDH genes in these tissues. In this case, primers were designed to the divergent 3′-untranslated regions so annealing temperatures compatible with the real-time PCR were possible. Despite several different designs, AroDH-4 primers were not suitable for real-time PCR, since they gave multiple products. The expression of each gene was measured in the arodh-1 background and it was expressed relative to the wild type for each tissue type. This analysis showed that, as expected, no transcript is produced downstream of the Mu insertion site in arodh-1 and, furthermore, that AroDH-2 and AroDH-3 expression is not increased in arodh-1 mutant tissues (not shown). Wild-type endosperm, embryo, and leaf RNAs were then used to address the relative expression levels of AroDH-2 and AroDH-3, in comparison with AroDH-1, and to determine the tissue specificity of each gene family member. In Fig. 5B the expression of AroDH-2 and AroDH-3 in each tissue relative to the AroDH-1 expression level for each tissue type is shown. In endosperm, AroDH-2 is expressed at 18% the level of AroDH-1, and AroDH-3 at 60% of that level. AroDH-2 has the highest relative expression of the three genes in the leaf tissue, and AroDH-3 has the highest relative expression of the three genes in the embryo tissue. In Fig. 5C the expression of each gene in embryo and leaf tissue relative to its own endosperm expression level is shown to demonstrate the tissue specificity of each gene. These data show that AroDH-1 is predominantly an endosperm-expressed gene, whereas AroDH-2 shows little tissue specificity, although it is most highly expressed in the leaf. AroDH-3 expression is biased towards the seed and shows a greater relative embryo expression than does AroDH-1.

Bottom Line: In plants, the amino acids tyrosine and phenylalanine are synthesized from arogenate by arogenate dehydrogenase and arogenate dehydratase, respectively, with the relative flux to each being tightly controlled.A Mutator insertion at an equivalent position in AroDH-3, the most closely related family member to AroDH-1, is also associated with opaque endosperm and stunted vegetative growth phenotypes.Overlapping but differential expression patterns as well as subtle mutant effects on the accumulation of tyrosine and phenylalanine in endosperm, embryo, and leaf tissues suggest that the functional redundancy of this gene family provides metabolic plasticity for the synthesis of these important amino acids. mto140/arodh-1 seeds shows a general reduction in zein storage protein accumulation and an elevated lysine phenotype typical of other opaque endosperm mutants, but it is distinct because it does not result from quantitative or qualitative defects in the accumulation of specific zeins but rather from a disruption in amino acid biosynthesis.

View Article: PubMed Central - PubMed

Affiliation: Center for Plant Science Innovation, University of Nebraska, 1901 Vine St., Lincoln, NE 68588, USA. dholding2@unl.edu

ABSTRACT
In plants, the amino acids tyrosine and phenylalanine are synthesized from arogenate by arogenate dehydrogenase and arogenate dehydratase, respectively, with the relative flux to each being tightly controlled. Here the characterization of a maize opaque endosperm mutant (mto140), which also shows retarded vegetative growth, is described The opaque phenotype co-segregates with a Mutator transposon insertion in an arogenate dehydrogenase gene (zmAroDH-1) and this led to the characterization of the four-member family of maize arogenate dehydrogenase genes (zmAroDH-1-zmAroDH-4) which share highly similar sequences. A Mutator insertion at an equivalent position in AroDH-3, the most closely related family member to AroDH-1, is also associated with opaque endosperm and stunted vegetative growth phenotypes. Overlapping but differential expression patterns as well as subtle mutant effects on the accumulation of tyrosine and phenylalanine in endosperm, embryo, and leaf tissues suggest that the functional redundancy of this gene family provides metabolic plasticity for the synthesis of these important amino acids. mto140/arodh-1 seeds shows a general reduction in zein storage protein accumulation and an elevated lysine phenotype typical of other opaque endosperm mutants, but it is distinct because it does not result from quantitative or qualitative defects in the accumulation of specific zeins but rather from a disruption in amino acid biosynthesis.

Show MeSH