LkARF7 and LkARF19 overexpression promote adventitious root formation in a heterologous poplar model by positively regulating LkBBM1

Abstract

Cuttage propagation involves adventitious root formation induced by auxin. In our previous study, Larix kaempferi BABY BOOM 1 (LkBBM1), which is known to regulate adventitious root formation, was affected by auxin. However, the relationship between LkBBM1 and auxin remains unclear. Auxin response factors (ARFs) are a class of important transcription factors in the auxin signaling pathway and modulate the expression of early auxin-responsive genes by binding to auxin response elements. In the present study, we identified 14 L. kaempferi ARFs (LkARFs), and found LkARF7 and LkARF19 bound to LkBBM1 promoter and enhanced its transcription using yeast one-hybrid, ChIP-qPCR, and dual-luciferase assays. In addition, the treatment with naphthalene acetic acid promoted the expression of LkARF7 and LkARF19. We also found that overexpression of these two genes in poplar promoted adventitious root formation. Furthermore, LkARF19 interacted with the DEAD-box ATP-dependent RNA helicase 53-like protein to form a heterodimer to regulate adventitious root formation. Altogether, our results reveal an additional regulatory mechanism underlying the control of adventitious root formation by auxin.

Fig. 1. Expression analysis of LkARF genes in larch.

a Heat map of the expression of 14 LkARF genes in different tissues based on qRT-PCR. AR, adventitious roots; S, stems; ST, stem tips; L, leaves. The heat map was made using the Omiscshare tools (http://omicshare.com/tools/) with parameters as default. b–d qRT-PCR analysis of LkARF6-3 (b), LkARF7 (c), LkARF19 (d), and LkBBM1 (e) in the basal stem sections (0.5 cm) during in vitro propagation. DAC days after cutting. The LaEF1A gene was used as the internal control gene. The error bars represent standard deviations from three biological replicates; *P < 0.05 and **P < 0.01, t test.

Fig. 2. Subcellular localization of LkARF7 and LkARF19.

Subcellular localization of LkARF7 and LkARF19 in tobacco leaf cells after tobacco leaf agroinfiltration. Scale bars, 30 μm.

Fig. 3. Overexpression of LkARF7 and LkARF19 promoted AR formation in 84 K poplar.

a, b AR phenotypes on 3-week-old control plants (Ctrl, transformed with empty vector), wild-type (WT), LkARF7-OE, and LkARF19-OE lines. Scale bars, 5 cm. c, d Average AR number (c) and total AR length (d) in the control plants and overexpression lines of 3-week-old seedlings. e Rooting rate of the control plants, WT, and overexpression lines within 11 days following cutting (n = 30 per line). DAC, days after cutting. Error bars represent standard deviations from three biological replicates; *P < 0.05 and **P < 0.01, t test.

Fig. 4. Overexpression of LkARF7 and LkARF19 promoted root growth in 84 K poplar.

a AR phenotypes on 8-week-old control plants (Ctrl), wild-type (WT), LkARF7-OE, and LkARF19-OE lines. Scale bars, 10 cm. b Fresh and c dry weight of roots in the control plants, WT, and overexpression lines. Error bars represent standard deviations from three biological replicates; *P < 0.05 and **P < 0.01 t test.

Fig. 5. LkARF7 and LkARF19 directly bind to the LkBBM1 promoter and activate its expression.

a Activation of LkBBM1 expression by transient overexpression of LkARF7 and LkARF19, respectively. The value from the empty vector (CK) was set to 1.0. b Yeast one-hybrid analysis of interactions between LkARF7/19 and the LkBBM1 promoter. P, Positive control (p53HIS2 + pGAD53T); N, Negative control (pHIS2-LkBBM1 promoter + pGADT7); LkARF7 (pHIS2-LkBBM1 promoter + pGADT7-LkARF7); LkARF19 (pHIS2-LkBBM1 promoter + pGADT7-LkARF19). DDO, SD/-Trp-Leu; TDO, SD/-Trp-Leu-His. c Diagram of the LkBBM1 promoter showing the relative positions of three auxin response elements (AuxRE) and one abscisic acid response element (ABRE). d Binding of LkARF7 and LkARF19 with the LkBBM1 promoter using ChIP-qPCR assay. The relative abundance of LkBBM1 promoter fragments in chromatin was determined by isolation from the 35S::LkARF7-flag, 35S::LkARF19-flag, and 35S::flag lines. e Activation of the LUC expression driven by the LkBBM1 promoter reporter when co-transformed with 35S::LkARF7 and 35S::LkARF19, respectively. 62SK + Luc-LkBBM1 promoter was used as the negative control (Ctrl), and its value was set to 1. f Representative images of the LUC expression driven by the LkBBM1 promoter reporter when co-transformed with 35S::LkARF7 and 35S::LkARF19, respectively. Red and white signals indicate strong binding capacity between effectors and reporters. Scale bars, 4 cm. Error bars represent standard deviations from four biological replicates; *P < 0.05 and **P < 0.01, t test.

Fig. 6. Interaction between LkARF19 and LkRH53.

a Yeast two-hybrid analysis of interaction between LkARF19 and LkRH53. N, negative control (pGBKT7-LamC + pGADT7-LargeT); P, positive control (pGBKT7-p53 + pGADT7-LargeT); LkRH53 (pGBKT7-LkARF19 + pGADT7-LkRH53). DDO, SD/-Trp-Leu; QDO, SD/-Trp-Leu-His-Ade; QDO/X, SD/-Trp-Leu-His-Ade + X-α-gal. b Bimolecular fluorescence complementation assay analysis of interaction between LkARF19 and LkRH53. LkARF19-cYFP + nYFP and cYFP + LkRH53-nYFP were used as negative controls. Scale bars, 30 μm. c qRT-PCR analysis of LkRH53 in the basal stem sections (0.5 cm) during cutting propagation. DAC, days after cutting. The value on DAC 0 was set to 1. d Overexpression of LkRH53 promoted AR formation in 84 K poplar. Scale bars, 3 cm. e Average AR number and total AR length of the control plants, WT, and overexpression lines in 2-week-old seedlings. Error bars represent standard deviations from three biological replicates; *P < 0.05 and **P < 0.01, t test.

Fig. 7. A proposed model of the role of LkARF7 and LkARF19 in AR formation.

The expression of LkARF7 and LkARF19 increases after the semi-lignified stem is cutoff. Auxin also promotes the expression of LkARF7 and LkARF19. LkARF7 and LkARF19 bind to AuxREs and promote LkBBM1 transcription to regulate AR formation. In addition, LkARF19 interacts with LkRH53 to form heterodimers to regulate AR formation.