Characterization of abnormal tissue in stressed Arabidopsis thaliana ago1 mutants

As an essential component of the micro RNA pathway, ARGONAUTE1 (AGO1) is a key regulator of critical developmental pathways and defense responses in Arabidopsis thaliana. We previously observed that ago1 mutant plants develop abnormal tissue growths on their embryonic leaves (cotyledons) when grown under stress. The abnormal tissue was not necrotic in appearance and showed phenotypic similarities with undifferentiated plant tissue. Our goal was to characterize this abnormal tissue and understand this unusual phenotype. We had two hypotheses: a) abnormal tissue growths were a result of miRNA misregulation that caused the cotyledon tissue to revert to a stem cell-like state or b) abnormal growths were a result of an upregulated hypersensitive defense response exaggerated by stress. At the morphological level, we used scanning electron microscopy, confocal microscopy, and bright field microscopy to compare abnormal to wild-type tissue. We found that the abnormal tissue originates from below the epidermis, that many abnormal cells have lost nuclei, and that many abnormal cells are dead and dying. At the molecular level, we used expression of several marker genes to distinguish between our two hypotheses. We found that ago1 cotyledons with abnormal tissue had significantly up-regulated defense markers, while markers of stem cell identity were similar to wild type cotyledons. We conclude that the abnormal tissue is likely a product of an upregulated hypersensitive response in ago1 that was further exaggerated by stress. Our results highlight that the miRNA pathway plays a critical role in buffering the effects of environmental stresses.