Enhanced Conjugation of Auxin by GH3 Enzymes Leads to Poor Adventitious Rooting in Carnation Stem Cuttings

Abstract

Commercial carnation (Dianthus caryophyllus) cultivars are vegetatively propagated from axillary stem cuttings through adventitious rooting; a process which is affected by complex interactions between nutrient and hormone levels and is strongly genotype-dependent. To deepen our understanding of the regulatory events controlling this process, we performed a comparative study of adventitious root (AR) formation in two carnation cultivars with contrasting rooting performance, "2101-02 MFR" and "2003 R 8", as well as in the reference cultivar "Master". We provided molecular evidence that localized auxin response in the stem cutting base was required for efficient adventitious rooting in this species, which was dynamically established by polar auxin transport from the leaves. In turn, the bad-rooting behavior of the "2003 R 8" cultivar was correlated with enhanced synthesis of indole-3-acetic acid conjugated to aspartic acid by GH3 proteins in the stem cutting base. Treatment of stem cuttings with a competitive inhibitor of GH3 enzyme activity significantly improved rooting of "2003 R 8". Our results allowed us to propose a working model where endogenous auxin homeostasis regulated by GH3 proteins accounts for the cultivar dependency of AR formation in carnation stem cuttings.

Keywords: Dianthus caryophyllus; IAA degradation; adventitious rooting; auxin homeostasis; auxin-conjugating enzymes; polar auxin transport; stem cuttings.

FIGURE 1

Cultivar-dependent adventitious rooting in carnation stem cuttings. (A) Time series of adventitious rooting in carnation stem cuttings grown in vitro. A representative sample in each cultivar was imaged between 15 and 29 days after planting. (B) The average lengths ± standard deviations of scanned root systems between 13 and 29 days are shown for the studied cultivars. Letters indicate significant differences (P < 0.05) between data points (n = 20). (C) Light micrographs from cross-sections of stem cutting basal regions at 0 and 54 h after planting. Black arrowhead indicates periclinal cell divisions in the cambium (ca). cc, cell clusters; pl: phloem; xl, xylem.

FIGURE 2

Auxin transport through the stem cutting base is required for adventitious root (AR) formation. (A) Representative images of adventitious rooting stages in carnation stem cuttings growing in soil plugs as defined previously (Birlanga et al., 2015). To visualize the entire root system, the soil substrate was carefully removed with pressurized water. (B) Graphic representations of rooting stage values in different carnation cultivars and treatments at 29 days, as described in Birlanga et al. (2015). Different letters indicate significant differences (P < 0.05) over sample means (cultivar × treatment). (C) Representative images of adventitious rooting in the studied carnation cultivars at 29 days treated with exogenous auxins or a polar auxin transport inhibitor (1-NOA). (D) Basipetal auxin transport parameters, transport rate (mm h^-1), and transport intensity (ng IAA h^-1) measured in stem cutting basal sections of the studied cultivars. Average ± standard deviation values are shown. Letters indicate significant differences (P < 0.05) over samples.

FIGURE 3

Differential auxin biosynthesis and auxin signaling in mature leaves at harvest. (A) Scheme of a carnation stem cutting leaf with indication of the two regions studied. (B) Indole-3-pyruvic acid (IPyA) and (C) indole-3-acetic acid (IAA) were measured in mature leaves of carnation stem cuttings at severance time. Average ± standard deviation values are shown. (D,F) RT-qPCR of the expression of selected transcripts related to (D,E) auxin biosynthesis or (F) auxin signaling in mature leaves of carnation stem cuttings at harvesting time. Bars indicate normalized expression levels ± standard deviation relative to the distal region of the leaf in the “Master” cultivar. Letters indicate significant differences between samples (P < 0.05).

FIGURE 4

Endogenous levels of several auxin derivatives in the stem cutting base during adventitious rooting and expression of selected genes. (A) IAA, (B) indole-3-acetyl-L-aspartic acid (IAA-Asp), and (C) 2-oxo-indole-3-acetic acid (oxIAA). Average ± standard deviation values are shown at selected time-points: at harvesting time (H; –23 h), during storage at low temperature (–15 h), at planting time (P; 0 h), and during rooting (6, 24, and 54 h). Asterisks indicate significant differences between samples (P < 0.05). (D–F) Real-time PCR quantification of the expression of (D) DcIAA19, (E) DcGH3.1, and (F) DcDAO1 relative to the –23 h dataset in the “2101–02 MFR” cultivar (not shown in the graph). Bars indicate normalized expression levels ± standard deviation. Letters indicate significant differences between samples (P < 0.05).

FIGURE 5

Auxin homeostasis at the stem cutting base is required for AR formation. (A) Graphic representations of rooting stage values in different carnation cultivars and treatments (n = 50). (B) Representative images of adventitious rooting in the studied carnation cultivars at 29 days treated with GH3 inhibitor (10 μM AIEP) or with mock. (C,D) The percentage of cuttings with roots (C) and the area of the scanned root system (D) at 29 days in rooted stem cuttings (n = 50). Dark- and light-colored bars represent data from mock- or AIEP-treated samples, respectively. Different letters indicate significant differences (P < 0.05) over sample means (cultivar × treatment).

FIGURE 6

Analysis of expression of transcripts related to auxin (A) and cytokinin (CK) (B) homeostasis during AR formation. Heat map drawing and clustering was done as described in the section “Materials and Methods”. Arrowheads indicate those genes mentioned in the text with differential expression at defined time-points in 2101–02 MFR (gray arrowheads) or “2003 R 8” (black arrowheads). Putative positive regulators are indicated in green, while negative regulators are shown in red.

FIGURE 7

An integrated model of AR formation in carnation stem cuttings. (A) Regulation of auxin homeostasis in stem cutting leaves. Auxin influx proteins (AUX1/LAXs) are depicted in pink; auxin efflux proteins (PIN1 and ABCB19) are shown in purple. Size and ploidy level of mesophyll cells varied between distal (small size and 2C–4C ploidy level) and proximal (large size and 4C–8C ploidy level) leaf regions. The site of auxin synthesis is depicted in light blue. (B) Differences in auxin homeostasis in selected carnation genotypes during rooting of stem cuttings. In the leaf diagrams, the size of the purple arrow indicates the magnitude of the PAT from the leaves and the blue intensity indicates the level of auxin synthesis. In the stem cutting base diagrams, the height of the colored cylinders (red, blue, yellow) indicates the amount of hormone. IAA levels were highest during storage and were quickly downregulated after planting and during rooting. Treatment with AIEP (a well-known inhibitor of GH3 enzymes) results in a higher auxin-to-CK ratio at planting time that enhanced rooting in “2003 R 8” cultivar. Exogenous IAA treatment also enhanced rooting in the “2003 R 8” cultivar.