Strategic distribution of protective proteins within bran layers of wheat protects the nutrient-rich endosperm

Abstract

Bran from bread wheat (Triticum aestivum 'Babbler') grain is composed of many outer layers of dead maternal tissues that overlie living aleurone cells. The dead cell layers function as a barrier resistant to degradation, whereas the aleurone layer is involved in mobilizing organic substrates in the endosperm during germination. We microdissected three defined bran fractions, outer layers (epidermis and hypodermis), intermediate fraction (cross cells, tube cells, testa, and nucellar tissue), and inner layer (aleurone cells), and used proteomics to identify their individual protein complements. All proteins of the outer layers were enzymes, whose function is to provide direct protection against pathogens or improve tissue strength. The more complex proteome of the intermediate layers suggests a greater diversity of function, including the inhibition of enzymes secreted by pathogens. The inner layer contains proteins involved in metabolism, as would be expected from live aleurone cells, but this layer also includes defense enzymes and inhibitors as well as 7S globulin (specific to this layer). Using immunofluorescence microscopy, oxalate oxidase was localized predominantly to the outer layers, xylanase inhibitor protein I to the xylan-rich nucellar layer of the intermediate fraction and pathogenesis-related protein 4 mainly to the aleurone. Activities of the water-extractable enzymes oxalate oxidase, peroxidase, and polyphenol oxidase were highest in the outer layers, whereas chitinase activity was found only in assays of whole grains. We conclude that the differential protein complements of each bran layer in wheat provide distinct lines of defense in protecting the embryo and nutrient-rich endosperm.

Figure 1.

Micrographs of the isolated bran fractions. A, Outer bran fraction (epidermis and hypodermis). B, Intermediate bran fraction (cross cells, tube cells, testa, and nucellar tissue). C, Detailed view of the individual layers in the intermediate fraction (Cc, cross cells; Nu, nucellar tissue; T, testa; Tc, tube cells). D, Aleurone cells. [See online article for color version of this figure.]

Figure 2.

2-DE gels of the inner bran layer (aleurone). The highlighted spots show the different EST classes of 7S globulin, with the EST GenBank gi numbers shown in the legend. The unhighlighted gels are shown in Supplemental Figures S5 and S6. Top gel, pI 4 to 7; bottom gel, pI 6 to 11.

Figure 3.

Summary of the major proteins identified in bran tissue fractions, supernatant from imbibed grain, and the outer tissue fraction. A, Table of major bran tissue and water-soluble proteins identified. B, Scanning electron micrograph of a cross-section of bran showing the bran tissue fractions.

Figure 4.

Fluorescence immunolocalization of defense proteins in bran cross-sections overlaid on differential interference contrast images of cross-sections (labeled). Dark inset overlays in images show fluorescence labeling without the differential interference contrast overlay. A, Control treated only with secondary antibody. B, OXO antibody. C, XIP-1 antibody. D, PR-4 antibody.

Figure 5.

Immunofluorescence localization of PR4, XIP-1, and OXO in wheat varieties Chara and Wedgetail. Images are arrayed in alternating rows of differential interference contrast overlaid with immunofluorescence image, followed by immunofluorescence-only image.

Figure 6.

Scanning electron micrograph of a cross-section of bran showing the bran tissue layers with corresponding Xyl and Ara contents. A, Outer (P, pericarp), intermediate (Cc, cross cells; Nu, nucellar tissue; T, testa [seed coat]; Tc, tube cells), and inner (Al, aleurone cells; E, endosperm) layers. B, Xyl and Ara (mol %) in corresponding bran tissue types (Parker et al., 2005).