Evaluation of zma-miR408 and its target genes function on maize (Zea mays) leaf growth response to cold stress by VIGS-based STTM approach.

miR408 is a conserved plant miRNA family that is known to regulate genes involved in copper metabolism. However, the function of miR408 in maize leaf growth regulation under cold stress isn't defined. In this study, endogenous maize miR408 was transiently silenced by using virus-induced gene silencing (VIGS) combined with short tandem target mimic (STTM) approaches. To this end, STTM-miR408a/b was designed, synthesized, and applied to maize seedlings. Subsequently, STTM-miR408a/b (STTM) and mock-treated (M) seedlings were subjected to cold stress (C) and the growth response of the seedlings was monitored. Finally, STTM-miR408a/b-treatment successfully downregulated the expression of endogenous mir408a/b and upregulated their putative targets Basic Blue Protein (BBP) and Blue Copper Protein (BCP) antagonistically in the STTM and STTM_C groups compared to M and M_C groups. On the other hand, their putative target Laccase (LAC22) gene was upregulated in the STTM group compared to the M group, but there were no significant expression differences between the M_C and STTM_C groups. The elongation rate of the STTM-miR408a/b-treated second and third leaves was reduced by 10% and 19% resulting in 19% and 11% shortening, respectively. Furthermore, the activity of catalase (CAT) and glutathione reductase (GR) was decreased by 57% in STTM, M_C, and STTM_C, and 29% and 28% in the M_C and STTM_C groups and ascorbate peroxidase (APX) was increased by 15% in M_C and STTM_C groups, respectively. These findings illuminated the maize leaf growth response to cold via regulation of expression of miR408 and its target genes and antioxidant system.