Taro raphide-associated proteins: Allergens and crystal growth

Abstract

Calcium oxalate raphide crystals are found in bundles in intravacuolar membrane chambers of specialized idioblasts cells of most plant families. Aroid raphides are proposed to cause acridity in crops such as taro (Colocasia esculenta (L.) Schott). Acridity is irritation that causes itchiness and pain when raw/insufficiently cooked tissues are eaten. Since raphides do not always cause acridity and since acridity can be inactivated by cooking and/or protease treatment, it is possible that a toxin or allergen-like compound is associated with the crystals. Using two-dimensional (2D) gel electrophoresis and mass spectrometry (MS) peptide sequencing of selected peptides from purified raphides and taro apex transcriptome sequencing, we showed the presence on the raphides of peptides normally associated with mitochrondria (ATP synthase), chloroplasts (chaperonin ~60 kDa), cytoplasm (actin, profilin), and vacuole (V-type ATPase) that indicates a multistage biocrystallation process ending with possible invagination of the tonoplast and addition of mucilage that may be derived from the Golgi. Actin might play a crucial role in the generation of the needle-like raphides. One of the five raphide profilins genes was highly expressed in the apex and had a 17-amino acid insert that significantly increased that profilin's antigenic epitope peak. A second profilin had a 2-amino acid insert and also had a greater B-cell epitope prediction. Taro profilins showed 83% to 92% similarity to known characterized profilins. Further, commercial allergen test strips for hazelnuts, where profilin is a secondary allergen, have potential for screening in a taro germplasm to reduce acridity and during food processing to avoid overcooking.

Keywords: actin; biomineralization; calcium oxalate; crystalloplastids; profilin; transvacuolar strands.

FIGURE 1

Gene expressed in the taro apex associated with the raphides predicted peptides from mass spectrometry (MS) sequencing and apex transcription analysis. (a) Raphide genes, bars topped with “A” predicted to allergens and (b) actin and actin‐related peptides in taro apex, bars marked with “R” were found in the raphide MS analysis. Other expressed cytoskeletal related genes are given in the supporting information Table S7. Mean + SE, n = 3

FIGURE 2

Taro apex genes expressed and potentially involved in (a) calcium metabolism, transport, Ca‐dependent kinases and Ca‐binding and (b) genes expressed in the apex with potential roles in oxalate metabolism. Mean + SE, n = 3

FIGURE 3

Gene network showing eight interconnected clusters of genes. Genes highlighted in red were those genes predicted to be on the mature raphides. Network genes included in the analysis were those associated with the raphides, cytoskeleton, oxalate metabolism, endosomal, vesicle, and membrane fusion and transport. The partial correlation coefficients were all greater than +0.99. The genes found in the dense gray central cluster are given in the supporting information Table S4

FIGURE 4

Clustal W Phylogram of the five predicted profilin genes from taro with selected profilins from other species some of which have not been reported to cause an allergenic reaction. Full gene alignment is given in the supporting information Table S5

FIGURE 5

Alignment of taro's five profilins showing the high antigenicity areas (Jespersen et al., 2017) using BepiPred‐2.0 trained on epitopes and nonepitope amino acids determined from crystal structures, with predicted B‐cell epitopes greater than a threshold of 0.5. B‐cell epitope showing the five sections of the profilin sequence with greater than the default threshold of 0.5 and increase in the epitope score of the second epitope when the 17 amino acids insert that occurred in EVM0003866

FIGURE 6

Dip test results to taro leaf extract on Neogen Hazelnut ELISA test strip. The variety with low acridity was compared with a variety with high acridity. The low acrid and water control show only two second control bands, while the highly acrid variety (99‐6) showed a faint third band that was consistently seen in a number of replications. n = 3.

FIGURE 7

Simplified model for raphide bundle synthesis in taro, based on the peptides found associated with purified raphides and transcription data presented and the changes in organelles and crystals at different stages of cattail (Typha augustifolia L.) raphide development (Kausch & Horner, 1983) and other references cited in the discussion. (a) Earliest stage where actin serves as a foundation for crystal biomineralization and growth though not necessarily the initial nucleation site, potentially in transvacuolar strands or the interface between vacuoles with mitochrondria (M) and crystalloplastids (CP) closely associated with the growing raphides; (b) Intermediate stage where an invagination of the vacuolar membrane that envelopes growing raphides and actin filament bundles with some mitochondria and crystalloplastids. Endoplasmic membranes may also be engulfed in this process. A microautophagy model is indicated in this figure; macroautophagy involving the formation of a phagosome is possible. (c) Final stages with completion of raphide growth where captured mitochrondria and crystalloplastids undergo lysis and some of the peptides released from these organelles being deposited on the raphides along with mucilage synthesis (MU) transferred in vesicles from the Golgi, with actin acting as a guide. Individual figure not to the same scale. Created with BioRender.com