Temporal and spatial expression of the major allergens in developing and germinating peanut seed

Abstract

Peanut (Arachis hypogaea) seed proteins Ara h 1, Ara h 2, and Ara h 3 are considered to be the major peanut allergens. However, little is known about their temporal and spatial expression during seed development and upon germination and seedling growth. In this study, transcript levels of the three major peanut allergen genes, ara h 1, ara h 2, and ara h 3, and their corresponding proteins were found in all cultivars. Expression patterns were heterogeneous depending on the specific peanut allergen gene and the cultivars tested. However, ara h 3 expression patterns among the cultivars were more variable than ara h 1 and ara h 2. Transcripts were tissue specific, observed in seeds, but not in leaves, flowers, or roots, and were undetectable during seed germination. In situ hybridizations and immunotissue prints revealed that both embryonic axes and cotyledons expressed the allergens. However, more ara h 1 and ara h 3 messenger RNA was detected in cotyledons relative to embryonic axes. Allergen polypeptide degradation patterns were different in embryonic axes compared with cotyledons during germination and seedling growth, with levels of Ara h 1 and Ara h 2 dramatically reduced compared to the Ara h 3 polypeptides in embryonic axes. These characterization studies of major peanut allergen genes and their corresponding seed storage proteins can provide the basic information needed for biochemical and molecular approaches to obtain a hypoallergenic peanut.

Figure 1.

Northern-blot analysis for the three major peanut allergen genes in 12 genotypes during seed maturation. The top segment for each genotype is ara h 1, ara h 2, or ara h 3 transcripts, as indicated. The bottom segment for each genotype is 25S rRNA transcript used as an internal control. 1, 2, 3, and 4 represent seed developmental stages from immature to mature.

Figure 2.

Expression of ara h 1 and ara h 2 during seed maturation. A, Transcript levels of ara h 1 or ara h 2 were analyzed by northern blots. B, Protein levels of Ara h 1 and Ara h 2 were analyzed by western blots. 1, 2, 3, and 4 represent the seed developmental stages of ‘Georgia Green.’

Figure 3.

Seed-specific expression of the three major peanut allergen genes. A, Northern blots for tissues including flowers (F), leaves (L), roots (R), and four seed developmental stages of ‘Georgia Green’ (1–4) for ara h 1, ara h 2, and ara h 3 genes. ara h 1, ara h 2, and ara h 3 indicates transcript signals, respectively. As a control, the transcript levels of 25S rRNA (25S) were used. B, Transcript level of ara h 3 was analyzed by RT-PCR from mRNA extracted from flowers (F), leaves (L), roots (R), and stage 4 seed of ‘Georgia Green’ (GG4). The transcript level of 18S rRNA (18S) was used as an internal control with an amplified band of 315 bp. A 1-kb DNA marker (M; Promega) was used as a size standard.

Figure 4.

Localization of ara h 1, ara h 2, and ara h 3 transcripts and Ara h 1 and Ara h 2 in seed by tissue print hybridization. A, Localization of transcripts of ara h 1, ara h 2, and ara h 3 in seed cross sections (stage 3). To verify the specificity of the signal from the antisense RNA probe, a sense RNA probe was used as a negative control. B, Longitudinal section of seed hybridized with anti-Ara h 1 and anti-Ara h 2 antibodies. Embryonic axis (E) and cotyledon (C) are indicated. As a control, seed section blots were stained with toluidine blue. [See online article for color version of this figure.]

Figure 5.

Tissue print immunoblotting for Ara h 1 and Ara h 2 in seed using peanut-specific IgE. Seed cross sections hybridized with human serum containing peanut-specific IgE for Ara h 1 (A) and Ara h 2 (B). Negative control was performed with human serum containing nonspecific IgE to peanut allergens. Embryonic axis (E) and cotyledon (C) are indicated. As a control, seed section blots were stained with toluidine blue. [See online article for color version of this figure.]

Figure 6.

Expression of the three major peanut allergen genes in embryonic axes and cotyledons. A, Northern blots were performed for ara h 1, ara h 2, and ara h 3 with mRNA extracted from embryonic axes (Em) and cotyledons (Co). The top segment is ara h 1, ara h 2, or ara h 3 transcripts as indicated. The lower segment is 25S rRNA transcript used as an internal control. Signal values obtained from each gene were normalized using the 25S rRNA signal value and analyzed. Units on the y axis: allergen transcripts/25S r RNA transcript. Each bar represents the sd. B and C, SDS-PAGE (B) and western blots (C) hybridized with anti-Ara h 1 and anti-Ara h 2 antibodies to proteins from embryonic axes (Em), cotyledons (Co), and stage 3 whole seed of ‘Georgia Green’ (GG). Protein size marker (M; Novagen) is indicated. [See online article for color version of this figure.]

Figure 7.

Expression of the three major allergen genes during peanut seed germination and seedling growth. A, Northern blots were performed for ara h 1, ara h 2, and ara h 3 during peanut seed germination and seedling growth. ara h 1, ara h 2, and ara h 3 indicate transcript signals, respectively. As a control, the transcript levels of 25S rRNA (25S) were used. B and C, SDS-PAGE (B) and western blots (C) for Ara h 1 and Ara h 2. Ara h 1, Ara h 2, and Ara h 3 polypeptides are indicated as the major allergens along with their corresponding sizes. 24, 48, 72, 96, and 144, Harvesting times after imbibition (hours). For the 0 time point, seeds were harvested after 20 min of imbibition. Protein size marker (M; Novagen) is indicated. [See online article for color version of this figure.]

Figure 8.

SDS-PAGE and western blots of embryonic axes and cotyledons during germination and seedling growth. Peanut seed proteins were isolated from embryonic axes (A), and cotyledons (B) and their polypeptide patterns are shown on SDS-PAGE. Western blots were performed with proteins from embryonic axes (C) and cotyledons (D) to detect levels of Ara h 1 and Ara h 2 with anti-Ara h 1 and anti-Ara h 2 polyclonal antibodies. 1, 4, 12, 24, 48, 72, 96, and 144, Harvesting times after imbibition (hours). For the 0 time point, seeds were harvested after 20 min of imbibition. Protein size marker (M; Novagen) is indicated.

Figure 9.

Ara h 1 levels in PBs during germination and seedling growth. Western blots of Ara h 1 were performed on PBs from embryonic axes (A) and cotyledons (B). Western blots were performed with proteins from the PB-fraction pellet and supernatant after PB isolation. 4, 12, 24, 48, 72, and 96, Harvesting times after imbibition (hours). For the 0 time point, seeds were harvested after 20 min of imbibition.