Triticeae Genomics and Genetics, 2025, Vol.16, No.2, 79-91 http://cropscipublisher.com/index.php/tgg 78 might be less active in seeds, and they also included an ER retention signal for the AMP which could affect how it’s partitioned in tissues. One interesting observation: they added an endoplasmic reticulum retention tag to the AMP protein (to help it accumulate in certain cell compartments), and they speculated that a lot of the AMP might be getting secreted to the extracellular space or stuck in cell walls. This was inferred because even though they found AMP in all tissues, it wasn’t accumulating to extremely high levels in any one tissue, and its presence in roots was very low (likely due to promoter activity and tissue distribution). In summary, both the chitinase and the AMP proteins were successfully produced in the transgenic barley plants - the Western blot and ELISA data confirmed their presence. The chitinase was quite abundant in the vegetative tissues (leaves and stems and also present in the ears), and the AMP was distributed throughout the plant (with evidence that it’s secreted outside cells). Importantly, the researchers did not detect any unusual new protein bands or major changes in the protein profiles of the transgenic plants besides the antifungal proteins. In other words, producing these new proteins didn’t drastically alter the plant’s normal protein makeup. This suggests that the barley’s cellular systems were handling the production of these foreign proteins without issue. 4.3 Tissue-specific expression pattern To further understand where and when these antifungal genes were active in the plant, the researchers examined the tissue-specific expression patterns in the transgenic barley. They used qRT-PCR to compare Chi and AMP gene transcript levels in different tissues: roots, stems, leaves, and ears (spikes). The Chi gene (under the Ubiquitin promoter) was highly expressed in the above-ground parts - leaves and stems had strong expression, and there was also considerable expression in the ears (including the glumes that surround the grain). The lowest expression for Chi was in the roots (Choi et al., 2003), which is consistent with the behavior of the Ubiquitin promoter (it drives expression in many tissues but often is a bit weaker in roots for monocots). For the AMP gene (under the Actin promoter), the qRT-PCR showed moderate expression in leaves, stems, and ears, but almost undetectable levels in roots. This again fits the known pattern of the rice Actin promoter, which is very active in green tissues and seeds but not much in roots. They also looked at the protein level distribution. Western blots on protein extracts from different tissues echoed the mRNA findings: the chitinase protein was readily detected in leaves and in the ear tissues, but barely or not at all in roots. The AMP protein was hard to detect by Western, but given the mRNA and the ELISA results, it’s inferred that the AMP is present in leaves, stems, and seeds, and minimal in roots. The limited root expression isn’t a big problem, though, because most of the devastating barley diseases (powdery mildew, rusts, Fusarium head blight) attack the leaves or the spikes, not the roots. To visualize where the chitinase protein was accumulating in the plant tissues, the team performed an immunohistochemical staining on sections of transgenic barley. They took thin cross-sections of leaves and other organs, applied the anti-chitinase antibody, and then a secondary antibody with a marker to show where binding occurred. The stained sections showed that chitinase was present in the leaf epidermis and around the vascular bundles (particularly at the sheath cells surrounding the vascular bundles). This suggests that the chitinase enzyme was being secreted to the cell walls in those areas, aligning with the fact that a signal peptide was used to secrete it. They also saw staining in parts of the spike, such as the glumes (the outer bracts of the spike) and the anthers/filaments of the flowers. This means that during the flowering stage, the antifungal chitinase protein was present in the reproductive organs, which could help protect the developing grain from Fusarium or other pathogens that infect during flowering. Putting it all together, the introduced antifungal gene exhibited a broad expression pattern in the transgenic barley - it was active in most tissues except the roots. Crucially, it covered the main infection sites: leaves (for foliar pathogens) and the ear (for spike pathogens). The lower expression in roots is not particularly concerning because root diseases were not the target here. And even with high expression in many tissues, the plant didn’t show any obvious tissue damage or abnormalities. There was no evidence that having lots of chitinase in the cell walls, for
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