The auxin receptor TIR1 homolog (PagFBL 1) regulates adventitious rooting through interactions with Aux/IAA28 in Populus

Abstract

Adventitious roots occur naturally in many species and can also be induced from explants of some tree species including Populus, providing an important means of clonal propagation. Auxin has been identified as playing a crucial role in adventitious root formation, but the associated molecular regulatory mechanisms need to be elucidated. In this study, we examined the role of PagFBL1, the hybrid poplar (Populus alba × P. glandulosa clone 84K) homolog of Arabidopsis auxin receptor TIR1, in adventitious root formation in poplar. Similar to the distribution pattern of auxin during initiation of adventitious roots, PagFBL1 expression was concentrated in the cambium and secondary phloem in stems during adventitious root induction and initiation phases, but decreased in emerging adventitious root primordia. Overexpressing PagFBL1 stimulated adventitious root formation and increased root biomass, while knock-down of PagFBL1 transcript levels delayed adventitious root formation and decreased root biomass. Transcriptome analyses of PagFBL1 overexpressing lines indicated that an extensive remodelling of gene expression was stimulated by auxin signalling pathway during early adventitious root formation. In addition, PagIAA28 was identified as downstream targets of PagFBL1. We propose that the PagFBL1-PagIAA28 module promotes adventitious rooting and could be targeted to improve Populus propagation by cuttings.

Keywords: PagFBL1; PagIAA28; adventitious root development; auxin signalling.

Figure 1

Expression patterns of PagFBL1 during AR formation. GUS staining of Pro _{Pag FBL 1} ::GUS leafy stems (a, c, e, g, i, k) and their transverse sections (b, d, f, h, j, l); the samples were collected at 0 day (a, b), 2 days (c, d), 3 days (e, f), 4 days (g, h) 5 days (i, j) and 6 days (h, l). Experiments were repeated three times for each, and the representative phenotypes are shown. Scale bars: (a, c, e, g, i, k) 1 mm; (b, d, f, h, j, l) 200 μm.

Figure 2

ARs from leafy stems of PagFBL1 overexpressed lines #4 and #18, knock‐down lines #2 and #12 and WT. (a–e) for #4 and #18: (a) the early stage of ARs. (b) Rooting rates as the percentage of leaf stem explants with emerged ARs. (c) Number of AR induced. (d) AR system from 5 months plants in soils. (e) The quantification of ARs from 5 months plants; (f–g) for #2 and #12: (f) the early stage of ARs. (g) Rooting rates. (h) Number of AR induced. (i) AR system from 2 months plants in soils. (j) The quantification of ARs from 2 months plants. Bars = 1 cm. The values are means ± SE of three replicates. Significant differences between WT and transgenic lines are indicated with asterisks (*P < 0.05 and **P < 0.01).

Figure 3

Venn diagrams showing the number of DEGs classified into groups of 0, 12, 24, 48 h after AR induction. (a) Up‐regulated genes from 12 h vs 0 h and 24 h vs 12 h. (b) Down‐regulated genes from 12 h vs 0 h and 24 h vs 12 h. (c) Up‐regulated genes from 24 h vs 12 h and 48 h vs 24 h. (d) Down‐regulated genes from 24 h vs 12 h and 48 h vs 24 h. (e) COG classification of DEGs in signal transduction mechanisms. (f) DEG percentages for major hormones in plant hormone signal transduction based on KEGG pathway.

Figure 4

Expression profiles of the genes (Table S2) related to auxin signalling pathways at different time points during AR formation by both qRT‐PCR and RNA‐Seq (fold change for FPKM).

Figure 5

Interactions of PagFBL1 and PagIAAs revealed by BiFC assay and LexA yeast two‐hybrid assay. (a) PagFBL1 and PagIAA 28.1 or 28.2 by BiFC assay. Bars = 12 μm. (b) PagFBL1 and PagIAA28.1 or 28.2 by LexA yeast two‐hybrid assay.