Antisense Oligonucleotides as a Gene-Silencing Strategy Regulating Cytosolic G6PDH in Hordeum vulgare

Antisense oligonucleotides (ASOs) are short, synthetic DNA fragments able to modulate gene expression. In this work, the cytosolic isoform of glucose-6-phosphate dehydrogenase (Cyt-G6PDH) was selected as a target for ASOs to induce transient gene silencing in barley (Hordeum vulgare). G6PDH is the most important enzyme of the oxidative pentose phosphate pathway (OPPP), supplying NADPH and regulating the entire cycle. Different ASOs were tested at different concentrations on the leaf surface. Their effects were evaluated by measuring enzymatic activity, gene expression, and protein abundance. Treatment with 30 µM ASOs for 6 h represented the optimal condition, inducing a 40-60% reduction in G6PDH total activity in barley leaves, with a specific decrease in the cytosolic isoform, as determined by DTT-sensitive enzymatic assays. ASOs designed on the main regulator of cyt-G6PDH, the shaggy-like kinase (SK11), led to an analogous effect in terms of G6PDH activity, confirming the involvement of HvSK11 in the regulation of cyt-G6PDH in barley. Consistently, qRT-PCR analyses showed that ASO treatment simulates an abiotic stress condition obtained by the down-regulation of Cyt-G6PDH. These results support the use of ASOs as a rapid and efficient method for the functional analysis of key metabolic regulators in plants to overcome complications in recalcitrant organisms or lethal genes.