Comprehensive Phytohormone Profiling of Kohlrabi during In Vitro Growth and Regeneration: The Interplay with Cytokinin and Sucrose

Abstract

The establishment of an efficient protocol for in vitro growth and regeneration of kohlrabi (Brassica oleracea var. gongylodes) allowed us to closely examine the phytohormone profiles of kohlrabi seedlings at four growth stages (T1-T4), additionally including the effects of cytokinins (CKs)-trans-zeatin (transZ) and thidiazuron (TDZ)-and high sucrose concentrations (6% and 9%). Resulting phytohormone profiles showed complex time-course patterns. At the T2 stage of control kohlrabi plantlets (with two emerged true leaves), levels of endogenous CK free bases and gibberellin GA₂₀ increased, while increases in jasmonic acid (JA), JA-isoleucine (JA-Ile), indole-3-acetic acid (IAA) and indole-3-acetamide (IAM) peaked later, at T3. At the same time, the content of most of the analyzed IAA metabolites decreased. Supplementing growth media with CK induced de novo formation of shoots, while both CK and sucrose treatments caused important changes in most of the phytohormone groups at each developmental stage, compared to control. Principal component analysis (PCA) showed that sucrose treatment, especially at 9%, had a stronger effect on the content of endogenous hormones than CK treatments. Correlation analysis showed that the dynamic balance between the levels of certain bioactive phytohormone forms and some of their metabolites could be lost or reversed at particular growth stages and under certain CK or sucrose treatments, with correlation values changing between strongly positive and strongly negative. Our results indicate that the kohlrabi phytohormonome is a highly dynamic system that changes greatly along the developmental time scale and also during de novo shoot formation, depending on exogenous factors such as the presence of growth regulators and different sucrose concentrations in the growth media, and that it interacts intensively with these factors to facilitate certain responses.

Keywords: cytokinin; in vitro development; kohlrabi; phytohormone profiling; shoot regeneration; sucrose.

Figure 1

Changes in endogenous levels (in pmol g⁻¹ FW) of indole-3-acetic acid (IAA) and related metabolites at four developmental stages (T1–T4) during in vitro growth of kohlrabi (cv. Vienna Purple) on media supplemented with 3(control)/6/9% sucrose or 2 mg L⁻¹ transZ/TDZ. (A) IAA; (B) indole-3-acetamide (IAM); (C) indole-3-acetonitrile (IAN); (D) IAA-aspartate (IAA-Asp). Results are expressed as mean ± SE (n = 3 independent biological replicates). Means marked with * are significantly different from control according to Student’s t-test (p < 0.05) at each developmental stage. transZ: trans-zeatin; TDZ: thidiazuron.

Figure 2

Changes in endogenous levels (in pmol g⁻¹ FW) of indole-3-acetic acid (IAA) metabolites and phenolic auxin analogues at four developmental stages (T1–T4) during in vitro growth of kohlrabi (cv. Vienna Purple) on media supplemented with 3(control)/6/9% sucrose or 2 mg L⁻¹ transZ/TDZ. (A) 2-oxindole-3-acetic acid (OxIAA); (B) oxo-IAA-glucose ester (OxIAA-GE); (C) phenylacetic acid (PAA); (D) benzoic acid (BzA). Results are expressed as mean ± SE (n = 3 independent biological replicates). Means marked with * are significantly different from control according to Student’s t-test (p < 0.05) at each developmental stage. transZ: trans-zeatin; TDZ: thidiazuron.

Figure 3

Pearson’s correlation coefficient (r) between the content of IAA and various auxin metabolites and/or analogues of kohlrabi (cv. Vienna Purple) seedlings treated with high sucrose concentration or cytokinins at four developmental stages (T1–T4).

Figure 4

Changes in endogenous levels (in pmol g⁻¹ FW) of free cytokinin bases at four developmental stages (T1–T4) during in vitro growth of kohlrabi (cv. Vienna Purple) on media supplemented with 3(control)/6/9% sucrose or 2 mg L⁻¹ transZ/TDZ. (A) trans-zeatin (transZ); (B) cis-zeatin (cisZ); (C) dihydrozeatin (DHZ); (D) N⁶-(Δ²-isopentenyl)adenine (iP). Results are expressed as mean ± SE (n = 3 independent biological replicates). Means marked with * are significantly different from control according to Student’s t-test (p < 0.05) at each developmental stage. transZ: trans-zeatin; TDZ: thidiazuron.

Figure 5

Relationship between endogenous auxin and CK free bases. Calculation of (A) ratio and (B) Pearson’s correlation coefficient (r) between the content of IAA and total CK free bases of kohlrabi (cv. Vienna Purple) seedlings treated with high sucrose concentration or cytokinins at four developmental stages (T1–T4). Results are expressed as mean ± SE (n = 3 independent biological replicates). Means of IAA/CK free base ratios marked with * are significantly different from control according to Student’s t-test (p < 0.05) at each developmental stage. transZ: trans-zeatin; TDZ: thidiazuron.

Figure 6

Changes in endogenous levels (in pmol g⁻¹ FW) of abscisic acid (ABA) and related metabolites at four developmental stages (T1–T4) during in vitro growth of kohlrabi (cv. Vienna Purple) on media supplemented with 3(control)/6/9% sucrose or 2 mg L⁻¹ transZ/TDZ. (A) ABA; (B) phaseic acid (PA); (C) dihydrophaseic acid (DPA); (D) ABA-glucose ester (ABA-GE); (E) 9-hydroxy-ABA (9OH-ABA). Results are expressed as mean ± SE (n = 3 independent biological replicates). Means marked with * are significantly different from control according to Student’s t-test (p < 0.05) at each developmental stage. transZ: trans-zeatin; TDZ: thidiazuron.

Figure 7

Pearson’s correlation coefficient (r) between content of (A) ABA and various ABA metabolites; (B) jasmonic acid (JA) and two JA metabolites; (C) JA and salicylic acid (SA) of kohlrabi (cv. Vienna Purple) seedlings treated with high sucrose concentration or cytokinins at four developmental stages (T1–T4).

Figure 8

Changes in endogenous levels (in pmol g⁻¹ FW) of jasmonic acid (JA) and related metabolites at four developmental stages (T1–T4) during in vitro growth of kohlrabi (cv. Vienna Purple) on media supplemented with 3(control)/6/9% sucrose or 2 mg L⁻¹ transZ/TDZ. (A) JA; (B) JA-isoleucine (JA-Ile); (C) cis-(+)-12-oxo-phytodienoic acid (cis-OPDA). Results are expressed as mean ± SE (n = 3 independent biological replicates). Means marked with * are significantly different from control according to Student’s t-test (p < 0.05) at each developmental stage. transZ: trans-zeatin; TDZ: thidiazuron.

Figure 9

Changes in endogenous levels (in pmol g⁻¹ FW) of (A) salicylic acid (SA); (B) 1-aminocyclopropane-1-carboxylic acid (ACC); and (C) gibberellin GA₂₀ at four developmental stages (T1–T4) during in vitro growth of kohlrabi (cv. Vienna Purple) on media supplemented with 3(control)/6/9% sucrose or 2 mg L⁻¹ transZ/TDZ. Results are expressed as mean ± SE (n = 3 independent biological replicates). Means marked with * are significantly different from control according to Student’s t-test (p < 0.05) at each developmental stage. transZ: trans-zeatin; TDZ: thidiazuron.

Figure 10

Principal component analysis (PCA) of the endogenous content of the phytohormone metabolites in kohlrabi (cv. Vienna Purple) during in vitro growth on media supplemented with 3(control)/6/9% sucrose or 2 mg L⁻¹ transZ/TDZ at four developmental stages: T1 (A); T2 (B); T3 (C); and T4 (D). The contributions of principal component 1 (PC1) and principal component 2 (PC2) to the overall variability of the measured data are indicated on the horizontal and vertical axes of the graphs, respectively. transZ: trans-zeatin; TDZ: thidiazuron.

Figure 11

Principal component analysis (PCA) of the endogenous content of the phytohormone metabolites at four developmental stages (T1–T4) during in vitro growth of kohlrabi (cv. Vienna Purple) on media supplemented with 3% sucrose (control) (A); 6% sucrose (B); 9% sucrose (C); 2 mg L⁻¹ transZ (D); and 2 mg L⁻¹ TDZ (E). The contributions of principal component 1 (PC1) and principal component 2 (PC2) to the overall variability of the measured data are indicated on the horizontal and vertical axes of the graphs, respectively. transZ: trans-zeatin; TDZ: thidiazuron.

Figure 12

Heat map showing the relative abundance of the analyzed metabolites on treatments with both cytokinin (transZ or TDZ at 2 mg L⁻¹) and high concentration of sucrose (6% or 9%) compared with the corresponding cytokinin-amended media with 3% sucrose. The ratios were calculated at four developmental stages (T1–T4) during in vitro growth of kohlrabi (cv. Vienna Purple). Additional arrows are provided for visual understanding of the results, and demonstrate arbitrarily set values (≥1; <1.1 and >0.9; ≤0.9); the color scale ranges from the minimum value (0) to the maximum value (21.6). */** The square boxes refer to two values (120.02 and 151.2, respectively) calculated above the set maximum; for interpretation reasons, these values are excluded when adjusting the color scale. transZ: trans-zeatin; TDZ: thidiazuron.