PagARF3.1 promotes adventitious root formation by repressing IPT-mediated cytokinin biosynthesis

Abstract

Adventitious root formation is a crucial developmental process in the clonal propagation of economically significant horticultural and woody species. This process is antagonistically regulated by auxin and cytokinin, yet the underlying molecular regulatory mechanisms remain poorly understood. In this study, we investigated the role of PagARF3.1, a homolog of Arabidopsis auxin response factor 3, in hybrid poplar (Populus alba × Populus glandulosa clone cv. '84K'), focusing on its involvement in adventitious root formation. GUS staining analysis revealed that PagARF3.1 was expressed in adventitious root tips, pericycle cells, early root primordia, and outgrowing roots. Knockdown of PagARF3.1 delayed adventitious root formation and reduced root biomass in transgenic plants, whereas overexpression of PagARF3.1 promoted earlier rooting and increased the number of adventitious roots. Real-time quantitative polymerase chain reaction analysis indicated that the expression levels of PagIPT5a and PagIPT5b were significantly elevated in PagARF3.1 RNAi lines and reduced in overexpression lines. Yeast one-hybrid assays and ChIP-PCR analysis demonstrated that PagARF3.1 directly binds to the promoter regions of PagIPT5a and PagIPT5b, thereby regulating their expression. These findings collectively demonstrate that PagARF3.1 acts as a positive regulator of adventitious root formation by repressing IPT-mediated cytokinin biosynthesis.

Keywords: Adventitious roots; Auxin signaling; Cytokinin; PagARF3.1; PagIPTs; Populus.

Figure 1

Expression patterns and subcellular localization of PagARF3.1. GUS-staining assays of PagARF3.1 in (a) 3-week-old sapling and (b)−(d) root. (e) The expression of PagARF3.1 in roots, leaf, and stem. (f) The nuclear localization of PagARF3.1.

Figure 2

Expression patterns of PagARF3.1 during AR formation. GUS staining of proPagARF3.1::GUS (a)−(d) leafy stems, and their (e)−(h) transverse sections; the samples were collected at (a), (e) 0 d, (b), (f) 3 d, (c), (g) 4 d, (d), (h) 6 d. Scale bars: (a)−(d) 1 mm; (e)−(g) 50 µm; (h), 200 µm

Figure 3

Manipulation of PagARF3.1 expression affects the AR development of transgenic poplar. (a) Representative images of the AR formation of WT and RNAi_1 and RNAi_15 after 7 d excision. (b) Representative images of the AR formation of WT and OE_B and OE_F after 5 d excision. Bars = 5 mm. (c) RT-qPCR analysis of PagARF3.1 transcripts in the WT and overexpression lines. (d) RT-qPCR analysis of PagARF3.1 transcripts in the wild type and RNAi lines. (e) Rooting rates of wild type, OE, and RNAi lines.

Figure 4

ARs from leafy stems of PagARF3.1 RNAi lines, OE lines, and WT. (a) AR systems of WT, OE, and RNAi line after 4 weeks of transfer to rooting induction medium. (b) AR system from 3 month old plants in soil. (c) Number of adventitious roots per cutting in WT, OE, and RNAi line. (d) The dry weight of ARs from 3 month old plants. Bars = 2 cm. Significant differences between WT and transgenic lines are indicated with asterisks. Significance analysis was performed by Student's t-test (** p < 0.01).

Figure 5

PagARF3.1 promotes AR formation via the cytokinin pathway. Comparison of AR formation from leaf explants between the WT and the overexpression line (#B, #F) of PagARF3.1 treated with (a) 0 µg/L 6-BA, (b) 0.5 µg/L 6-BA. (c) 1.5 µg/L 6-BA. Comparison of (d) AR formation frequency, and (e) the numbers of AR. Significance analysis was performed by Student's t-test (* p < 0.05 and ** p < 0.01). Bar = 2 cm.

Figure 6

PagARF3.1 negatively regulates PagIPT5a and PagIPT5b expression. (a)−(c) RT-qPCR analysis of PagARF3.1, PagIPT5a, and PagIPT5b during AR induction. (d), (e) PagIPT5a and PagIPT5b expression levels in PagARF3.1 RNAi and OE lines.

Figure 7

PagARF3.1 directly binds to the promoters of PagIPT5a and PagIPT5b. (a) Yeast one-hybrid analysis, and (b) ChIP-PCR analyses showed that PagARF3.1 can directly bind to the PagIPT5a and PagIPT5b promoters. Input, chromatin preparation before immunoprecipitation. Mock, immunoprecipitated without anti-FLAG antibody. Anti, immunoprecipitated with anti-FLAG antibody.