Distribution of indole-3-acetic acid in Petunia hybrida shoot tip cuttings and relationship between auxin transport, carbohydrate metabolism and adventitious root formation

Abstract

To determine the contribution of polar auxin transport (PAT) to auxin accumulation and to adventitious root (AR) formation in the stem base of Petunia hybrida shoot tip cuttings, the level of indole-3-acetic acid (IAA) was monitored in non-treated cuttings and cuttings treated with the auxin transport blocker naphthylphthalamic acid (NPA) and was complemented with precise anatomical studies. The temporal course of carbohydrates, amino acids and activities of controlling enzymes was also investigated. Analysis of initial spatial IAA distribution in the cuttings revealed that approximately 40 and 10 % of the total IAA pool was present in the leaves and the stem base as rooting zone, respectively. A negative correlation existed between leaf size and IAA concentration. After excision of cuttings, IAA showed an early increase in the stem base with two peaks at 2 and 24 h post excision and, thereafter, a decline to low levels. This was mirrored by the expression pattern of the auxin-responsive GH3 gene. NPA treatment completely suppressed the 24-h peak of IAA and severely inhibited root formation. It also reduced activities of cell wall and vacuolar invertases in the early phase of AR formation and inhibited the rise of activities of glucose-6-phosphate dehydrogenase and phosphofructokinase during later stages. We propose a model in which spontaneous AR formation in Petunia cuttings is dependent on PAT and on the resulting 24-h peak of IAA in the rooting zone, where it induces early cellular events and also stimulates sink establishment. Subsequent root development stimulates glycolysis and the pentose phosphate pathway.

Fig. 1

a Different sections of an excised leafy cutting of P. hybrida collected for IAA analysis. b IAA concentrations and c IAA contents in leaves (L) of different positions. d Relationship between leaf fresh mass and IAA concentration. SA shoot apex including smallest enclosing leaves; SM medium shoot position (1.5–2.5 cm from the basal end); SB stem base (0–0.5 cm). Mean and SE from five individual cuttings, columns which do not share a common letter are significantly different (P ≤ 0.05, Kruskal–Wallis test, n = 5). log log⁻¹ plot of IAA concentrations versus leaf mass of 20 individual leaves (L1–L4) from five individual cuttings

Fig. 2

a IAA concentrations in transversal sections of L6 leaf. b IAA concentrations in different stem positions. c Total IAA pool sizes in different cutting parts of P. hybrida. L leaves of different positions, as shown in Fig. 1a. US complete upper stem above the stem base (SB); tip, leaf section 0–1.5 cm; centre, leaf section >1.5 cm and ≤3 cm; base, leaf section >3 cm. In a and b, mean and SE from five individual cuttings. Columns which do not share a common letter are significantly different (P ≤ 0.05, Kruskal–Wallis test, n = 5). Dithered lines indicate the mean IAA level in the complete lamina and shoot. IAA contents in c were calculated from IAA concentrations as illustrated in Fig. 1b and Fig. 2b (mean value of the three stem sections) and as estimated for L5 (14 pmol g⁻¹ FW as mean value of L4 and L6) using the following fresh masses (mg): L1 (42), L2 (79), L3 (174), L4 (239), L5 (390), L6 (473), SB (65), US (290)

Fig. 3

Picture of overall rooting of representative control (left) and NPA-treated (50 µM) cuttings of Petunia hybrida ‘Mitchell’ at 14 dpe. The background was digitally blackened

Fig. 4

Light microscopy of stem base of P. hybrida cuttings during rooting under non-treated and NPA-treated conditions. In all micrographs, semi-thin cross-sections from 1 to 4 mm above the excision site are shown. a, b 24 hpe control cuttings. c, d 24 hpe NPA-treated cuttings. e, f 72 hpe control cuttings. g, h 72 hpe NPA-treated cuttings. i, j 144 hpe control cuttings. k, l 144 hpe NPA-treated cuttings. m, n 192 hpe control cuttings. o, p 192 hpe NPA-treated cuttings. Ca cambium, Co cortex, M meristem of AR, Md meristemoid of AR, Pi pith, RP primordia of AR, Vt vascular tissue, hpe hours post excision

Fig. 5

Temporal course of IAA concentrations in the lowest leaf (L6, a) and the basal stem (0.5 cm, b) of cuttings of P. hybrida during rooting under non-treated (solid line) and NPA-treated (dashed line) conditions (n = 5, each sample from two individual cuttings). Asterisks indicate a significant effect of the NPA treatment at the specified time after excision of cuttings (Mann–Whitney U test, P ≤ 0.05). c Transcript accumulation of auxin-responsive GH3 gene in the basal stem of P. hybrida during rooting under standard (non-treated) conditions. Northern blot analysis was performed with 20 g RNA per sample, and separation on a 1.5 % (w/v) formaldehyde agarose gel. After transfer of RNA to a nitrocellulose membrane, it was hybridized to radioactively labelled cDNA fragments of the corresponding gene. The below picture shows the loading control of rRNA, the ratio of intensity of 28S RNA to 18S RNA (for total RNA) is 1:1

Fig. 6

Alterations in the enzyme activities involved in sucrose degradation, glycolysis and pentose phosphate pathways in the basal stem of P. hybrida during rooting under non-treated (solid line) and NPA-treated (dashed line) conditions. a Cell wall invertase, b cytosolic invertase, c vacuolar invertase, d glucose-6-phosphate dehydrogenase, e phosphofructokinase. Each value is represented by the mean of five independent replicates ± SE. Asterisks indicate a significant effect of the NPA treatment at the specified time after excision of cuttings (t test, P ≤ 0.05)

Fig. 7

Concentrations of soluble sugars in the basal stem of P. hybrida during rooting under non-treated (solid line) and NPA-treated (dashed line) conditions. a Glucose, b fructose, c sucrose. Each value is represented by the mean of five independent replicates ± SE. Asterisks indicate a significant effect of the NPA treatment at the specified time after excision of cuttings (t test, P ≤ 0.05)

Fig. 8

Concentrations of amino acids in the basal stem of P. hybrida during rooting under non-treated (solid line) and NPA-treated (dashed line) conditions. a Total amino acids, b glutamate, c glutamine, d aspartate, e asparagine, f proline. Each value is represented by the mean of five independent replicates ± SE. Asterisks indicate a significant effect of the NPA treatment at the specified time after excision of cuttings (t test, P ≤ 0.05)

Fig. 9

A postulated model of relationship between PAT, auxin accumulation, primary metabolism and AR formation in Petunia cuttings in response to excision from the donor plant. Red solid arrows indicate processes that are dependent on PAT and are correlated with the resulting auxin peak at 24 hpe in the rooting zone. Red dashed arrows indicate additional processes which are hypothetically depending on PAT as supported by data of the present study and of Ahkami et al. (2009). Thin black arrows with question mark show possible interrelations between GH3 induction, auxin metabolism and cellular events of AR formation. Further details regarding different stages and phases of AR formation in petunia cuttings are described in Ahkami et al. (2009). PAT polar auxin transport, CW invertase cell wall invertase, PFK phosphofructokinase, Glc6PDH glucose-6-phosphate dehydrogenase, hpe hours post excision