Balancing of sulfur storage in maize seed

Abstract

Background: A balanced composition of amino acids in seed flour is critical because of the demand on essential amino acids for nutrition. However, seed proteins in cereals like maize, the crop with the highest yield, are low in lysine, tryptophan, and methionine. Although supplementation with legumes like soybean can compensate lysine deficiency, both crops are also relatively low in methionine. Therefore, understanding the mechanism of methionine accumulation in the seed could be a basis for breeding cultivars with superior nutritional quality.

Results: In maize (Zea mays), the 22- and 19-kDa α-zeins are the most prominent storage proteins, nearly devoid of lysine and methionine. Although silencing synthesis of these proteins through RNA interference (RNAi) raises lysine levels in the seed, it fails to do so for methionine. Computational analysis of annotated gene models suggests that about 57% of all proteins exhibit a lysine content of more than 4%, whereas the percentage of proteins with methionine above 4% is only around 8%. To compensate for this low representation, maize seeds produce specialized storage proteins, the 15-kDa β-, 18-kDa and 10-kDa δ-zeins, rich in methionine. However, they are expressed at variant levels in different inbred lines. A654, an inbred with null δ-zein alleles, methionine levels are significantly lower than when the two intact δ-zein alleles are introgressed. Further silencing of β-zein results in dramatic reduction in methionine levels, indicating that β- and δ-zeins are the main sink of methionine in maize seed. Overexpression of the 10-kDa δ-zein can increase the methionine level, but protein analysis by SDS-PAGE shows that the increased methionine levels occur at least in part at the expense of cysteines present in β- and γ-zeins. The reverse is true when β- and γ-zein expression is silenced through RNAi, then 10-kDa δ-zein accumulates to higher levels.

Conclusions: Because methionine receives the sulfur moiety from cysteine, it appears that when seed protein synthesis of cysteine-rich proteins is blocked, the synthesis of methionine-rich seed proteins is induced, probably at the translational level. The same is true, when methionine-rich proteins are overexpressed, synthesis of cysteine-rich proteins is reduced, probably also at the translational level. Although we only hypothesize a translational control of protein synthesis at this time, there are well known paradigms of how amino acid concentration can play a role in differential gene expression. The latter we think is largely controlled by the flux of reduced sulfur during plant growth.

Figure 1

Classification of maize storage proteins. Protein isolation and gel electrophoresis are described under Methods.

Figure 2

Zein accumulation pattern in inbred A654 and its derivative lines with intact δ-zein alleles, βRNAi, γRNAi or both RNAis. Protein from 500 μg of maize flour was loaded in each lane. M, protein markers from top to bottom being 37, 25, 20, 15 and 10 kDa. The size of each zein band is indicated with numbers in the “kDa” column.

Figure 3

Protein accumulation pattern of seeds from the cross of B73 xP6z1CRNAi. Lanes 1–3, vitreous kernels with genotype B73/-; −/−; lanes 4–6, opaque kernels with genotype B73/-; P6z1RNAi/-. Zein and non-zein accumulation patterns of B73/-; −/− and B73/-; P6z1RNAi/- were analyzed by 15% SDS-PAGE. Protein from 500 μg of maize flour was loaded in each lane. M, protein markers from top to bottom being 250, 150, 100, 75, 50, 37, 25, 20, 15 and 10 kDa. The size of each zein band is indicated with numbers in the “kDa” column.

Figure 4

Zein accumulation patterns of Hi-Met transgenic seeds.A, Hi-Met transgenic seeds in lane 2 and 4 accumulate significantly higher amounts of the 10-kDa δ-zein, but their β- and γ-zeins are lower than non-transgenic controls in lanes 1 and 4. B, linkage analysis of the Hi-Met transgene and the suppression of β- and γ-zeins in immature seeds from Hi-Met/- x B73 at 18 DAP. The kernels inheriting the Hi-Met transgene expressing higher level of the 10-kDa δ-zein are marked with “T.” C, as described in B, but seeds are at maturity. D, linkage analysis of the Hi-Met transgene and the suppression of β- and γ-zeins in the progeny from the cross of (Met/-; z1CRNAi/-) x (Hi-II B x A). The kernels inheriting the Hi-Met transgene expressing higher level of the 10-kDa δ-zein are marked red. M, protein markers from top to bottom being 37, 25, 20, 15 and 10 kDa. The size of each zein band is indicated with numbers in the “kDa” column.

Figure 5

Increased accumulation of the 10-kDa δ-zein in transgenic RNAi seeds.A, Comparison of the 10-kDa δ-zein level in non-transgenic control, βRNAi, γRNAi and βγRNAi in Hi-II background. B, linkage analysis of the 10-kDa δ-zein level and RNAis in seeds from the cross of B73 x γRNAi/-; βRNAi/-. C, linkage analysis of the 10-kDa δ- zein level and RNAis in seeds from the cross of Mo17 x γRNAi/-; βRNAi/-. Kernel genotypes are marked with different symbols. M, protein markers from top to bottom being 37, 25, 20, 15 and 10 kDa. The size of each zein band is indicated with numbers in the “kDa” column.

Figure 6

Pathways for sulfate reduction and synthesis of cysteine and methionine. The essential amino acids lysine, threonine, isoleucine and methionine are synthesized from the same precursor aspartic acid. Methionine is a sulfur-containing amino acid, in which the sulfur moiety is transferred from cysteine by CGS. The sulfide in cysteine is reduced from sulfate absorbed from the soil by three steps as shown in the diagram.