Tobacco TTG2 regulates vegetative growth and seed production via the predominant role of ARF8 in cooperation with ARF17 and ARF19

Jun Ge, Baoyan Li, Dan Shen, Junyi Xie, Juying Long, Hansong Dong

Plant TRANSPARENT TESTA GLABRA (TTG) proteins regulate various developmental activities via the auxin signaling pathway. Recently, we elucidated the developmental role of tobacco (Nicotiana tabacum L.) NtTTG2 in association with 12 genes that putatively encode AUXIN RESPONSIVE FACTOR (ARF) proteins, including NtARF8, NtARF17, and NtARF19. Here we show that NtTTG2 regulates tobacco growth and development by involving the NtARF8, NtARF17, and NtARF19 genes, with the NtARF8 gene playing a predominant contribution. Independent silencing of the NtARF8 gene more strongly repressed tobacco growth than silencing the NtARF17 or NtARF19 gene and more effectively eradicated the growth enhancement effect of NtTTG2 overexpression. In contrast, plant growth was not affected by silencing additional nine NtTTG2-regulated NtARF genes. In double and triple gene silencing combinations, silencing the NtARF8 gene was more effective than silencing the NtARF17 or NtARF19 gene to repress growth as well as nullify growth enhancement. Therefore, the NtARF8 predominantly cooperated with the NtARF17 and NtAFR19 of the NtTTG2 functional pathway. NtARF8 also contributed to NtTTG2-regulated seed production as concurrent NtTTG2 and NtARF8 overexpression played a synergistic role in seed production quantity, whereas concurrent silencing of both genes caused more severe seed abortion than single gene silencing. In plant cells, the NtTTG2 protein facilitated the nuclear import of NtARF8 as well as increased its function as a transcription activator. NtARF8 is an integral component of the NtTTG2 functional pathway, which regulates tobacco growth and development.