Generation and Characterization of a Genetically Modified Zea mays Line with a Knockdown of Hypoxia-Dependent...

Hypoxia-dependent microRNAs play an important role in orchestrating a plant's response to low-oxygen stress. To assess the regulatory mechanisms of the adaptive response of maize (Zea mays L.) to hypoxia, an antisense sequence was developed, and the short tandem target mimic (STTM) system was used to induce the loss of function of the mature microRNA775A (miR775a) in maize. A recombinant binary vector pBI121 cloned in E. coli cells containing the antisense sequence anti-miR775A to maize miR775A was acquired to create a line of modified A. tumefaciens EHA105. Using the puncturing method on soaked seeds, maize plants with an active anti-miR775A construct were obtained, as evidenced by a decrease of more than 10-fold in mature miR775A content and by developmental changes in the seedlings. The size of seedlings of the maize knockdown line was almost twice smaller than that of the wild-type (WT) plants. An assessment of the effects of hypoxic conditions induced by flooding of 14-day-old maize plants revealed differences in the expression and activity of several enzymes between WT and knockdown plants. The reduced miR775A levels led to a 2.1-fold drop in pyruvate levels, which resulted in decreased pyruvate kinase, pyruvate dehydrogenase, and lactate dehydrogenase activities as compared to WT plants. A decrease in miR775A content in the maize knockdown cell line also affected the function of mitochondrial and extramitochondrial isoenzymes of citrate synthase, aconitase, and fumarase under hypoxic conditions.