Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

Summary

Adventitious shoots can be induced on internodal segments of ipecac without phytohormone treatment. To evaluate phytohormone dynamics during adventitious shoot formation, we measured endogenous auxin and cytokinin in internodal segments by LC-MS/MS.

Abstract

Adventitious shoot formation is an important technique for the propagation of economically important crops and for the regeneration of transgenic plants. Phytohormone treatment is required for the induction of adventitious shoots in most species. Whether adventitious shoots can be induced is determined by the balance between auxin and cytokinin (CK) levels. Much effort goes into determining optimum concentrations and combinations of phytohormones in each tissue used as explants and in each plant species. In ipecac, however, adventitious shoots can be induced on internodal segments in culture medium without phytohormone treatment. This allows the inherent plasticity of ipecac for cell differentiation to be evaluated. To induce adventitious shoots in ipecac, we cultured internodal segments at 24 °C under 15 µmol m⁻² s⁻¹ of light in a 14-h light/10-h dark cycle on phytohormone-free B5 medium solidified with 0.2% gellan gum for 5 weeks. To investigate phytohormone dynamics during adventitious shoot formation, we measured endogenous indole-3-acetic acid and CKs in the segments by liquid chromatography-tandem mass spectrometry LC-MS/MS. This method allows analysis of endogenous indole-3-acetic acid and CKs levels in a simple manner. It can be applied to investigate the dynamics of endogenous auxin and CK during organogenesis in other plant species.

Introduction

Gottlieb Haberlandt (1854-1945) proposed the concept of "totipotency", by which plant cells can divide, differentiate, and regenerate whole plants even after their prior differentiation into specific cell types in mature plants¹. In tissue culture, whether plant regeneration can be induced or not is determined by the combination and concentration of exogenously applied phytohormones in the growth medium. Skoog and Miller found that adventitious shoots could be induced from tobacco callus on culture medium containing a high ratio of CKs to auxins, whereas adventitious roots could be induced on medium containing a low ratio². Since that finding, tissue culture has been widely used for the propagation of economically important crops and for the regeneration of transgenic plants³. Adventitious shoots can be induced from tissues other than shoot apical meristem, such as leaves, roots, and internodes. Phytohormone treatment is required for the induction of adventitious shoots in most plant species. However, the optimum concentrations and combinations differ by species and among tissues used as explants. Thus, much effort goes into determining the optimum concentrations and combinations of phytohormones for experiments.

Carapichea ipecacuanha (Brot.) L. Andersson (ipecac) is a medicinal plant that contains alkaloids such as emetine and cephaeline, mainly in the roots⁴. Root extracts are used as an expectorant, an emetic, and an amoebicide⁵. Although ipecac grows naturally in the tropical rainforests of Brazil, it is reluctant to set seeds in culture, and the germination rate decreases during seed storage in Japan, with its colder climate⁶. Instead, it is propagated by tissue culture, in which adventitious shoot formation on internodes is the most efficient method^7,8. Interestingly, adventitious shoots can be induced in this species without phytohormone treatment⁸.

Adventitious shoots are formed on the epidermis in the apical region of internodal segments without callusing, but not in the basal region⁹. This difference indicates tissue polarity in internodal segments, which is probably under phytohormonal regulation. The ipecac culture system allows a unique opportunity to analyze changes in endogenous phytohormone levels during adventitious shoot formation. Here we introduce our method for the analysis of the endogenous levels of one auxin (indole-3-acetic acid (IAA)) and four CKs (isopentenyl adenine (iP), isopentenyl adenine riboside (iPR), trans-zeatin (tZ), and trans-zeatin riboside (tZR)) in internodal segments through the use of LC-MS/MS.

Protocol

Note: Ipecac (C. ipecacuanha) was used in this study because it facilitates the analysis of endogenous phytohormones.

1. Growth Conditions to Induce Adventitious Shoots of Ipecac

1. Prepare phytohormone-free B5 medium adjusted to pH 5.7¹⁰, and add 0.2% gellan gum. Sterilize by autoclaving.
2. Pour 25 mL of the autoclaved medium into a sterile Petri dish (90 mm × 20 mm).
3. Cut 8-mm internodal segments of ipecac plantlets using a surgical scalpel with a blade No. 22 on a sterile acrylic plate, and place on the medium (Figure 1).
4. Culture on phytohormone-free B5 medium at 24 °C under 15 µmol m⁻² s⁻¹ of light in a 14-h light/10-h dark cycle for 5 weeks.
5. Identify regions I (apical) to IV (basal) in each segment (Figure 1B). Count the number of adventitious shoots > 0.3 mm length in each region under a microscope once a week.

2. Extraction and Purification of Phytohormones

1. Put a 5-mm zirconia bead in each of the four 2-mL sample tubes.
2. Cut the segments into regions I to IV (each 2 mm in length) using a surgical scalpel on a sterile acrylic plate.
3. Collect eight segments of each region in separate sample tubes (10-30 mg fresh weight).
4. Weigh, and then freeze the samples in liquid nitrogen.
5. Crush the frozen samples using a bead-based homogenizer.
6. Suspend the crushed samples in 1 mL acetonitrile containing 500 pg of each internal standard of auxin and CKs (d₅-IAA, d₅-tZ, d₅-tZR, d₆-iP, d₆-iPR) using a vortex mixer.
7. Hold at 4 °C for 1 h, then centrifuge at 3,500 × g for 5 min at room temperature.
8. Wash the pellet in 80% (v/v) acetonitrile containing 1% (v/v) acetic acid. Centrifuge again at 3,500 × g for 5 min at room temperature. Combine the supernatants (from steps 2.7 and 2.8) in a disposable glass tube.
9. Add 600 µL water containing 1% (v/v) acetic acid to each combined supernatant, and evaporate the acetonitrile using a vacuum concentrator.
10. Equilibrate hydrophilic-lipophilic-balanced (HLB) column cartridges by applying 1 mL each of acetonitrile, methanol, and water containing 1% (v/v) acetic acid.
11. Apply one sample solution per equilibrated HLB cartridge.
12. Wash the cartridges with 1 mL water containing 1% (v/v) acetic acid.
13. Elute all hormones with 2 mL 80% (v/v) acetonitrile containing 1% (v/v) acetic acid in a glass tube.
14. Evaporate the acetonitrile in the eluate to obtain extract in water containing 1% (v/v) acetic acid using a vacuum concentrator.
    NOTE: Do not dry this completely.
15. Equilibrate mixed-mode, strong cation-exchange (MCX) column cartridges by applying 1 mL acetonitrile, 1 mL methanol, 0.5 mL 0.1 M HCl, and 1 mL water containing 1% (v/v) acetic acid.
16. Apply one sample solution per equilibrated MCX cartridge.
17. Wash the cartridges with 1 mL water containing 1% (v/v) acetic acid.
18. Elute IAA with 2 mL 30% (v/v) acetonitrile containing 1% (v/v) acetic acid in a glass tube.
19. Wash the cartridges with 2 mL 80% (v/v) acetonitrile containing 1% (v/v) acetic acid.
20. Wash the cartridges with 2 mL water and 1 mL water containing 5% aqueous ammonia.
21. Elute the CKs with 2 mL 60% (v/v) acetonitrile containing 5% aqueous ammonia in a glass tube.
22. Evaporate the solvent of each hormone fraction using a vacuum concentrator and store at −30 °C until the LC-MS/MS analysis.

3. LC-MS/MS Analysis of IAA and CKs

1. Dissolve each hormone extract in a tube with 600 µL of methanol and transfer the solution to a screw neck total recovery vial. Evaporate the solvent using a vacuum concentrator.
2. Dissolve the IAA fraction in 20 µL 30% (v/v) acetonitrile, and the CK fractions in 20 µL water containing 1% (v/v) acetic acid in screw neck total recovery vials.
3. Analyze the samples in positive ion mode on a triple-quadrupole MS system equipped with an HPLC system.
   NOTE: We set the HPLC conditions as listed in Table 1.
   1. For the IAA elution, use a binary gradient of 5%-50% solvent B over 7 min, then increase by 98% solvent B and hold for 1 min, and then equilibrate for 2 min at 5% solvent B by nest injection.
   2. For the CK elution, use a binary gradient of 2%-40% solvent B over 5 min, 40%-70% solvent B in 7 min, then increase by 95% solvent B and hold for 1 min, and then equilibrate for 2 min at 2% solvent B by nest injection.
   3. Set the electrospray ionization (ESI)-MS parameters of ion source as listed in Table 2.
4. Use the multiple reaction monitoring (MRM) transition for quantification of each analyte listed in Table 3.
5. Quantify endogenous IAA and CK levels against a standard curve of the ratio of unlabeled to deuterium-labeled standards.

Results

At the 1^st week, no adventitious shoots had formed. At the 2^nd week, small shoots appeared. At the 3^rd and 4^th weeks, the number of shoots increased mostly in the apical regions (I and II) (Figure 2A). At the 5^th week, the number of shoots was approximately 7 in region I and 5 in region II (Figure 2B). In contrast, only a few shoots were form...

Discussion

To identify the distribution of phytohormones involved in organogenesis, it is important to use plant materials in which organogenesis can be observed on phytohormone-free medium, because when phytohormones are exogenously applied to explants for inducing shoots or roots, they affect the whole explant, making it difficult to evaluate the inherent plasticity of plants in cell differentiation and organogenesis. Adventitious shoots can be induced on phytohormone-free culture media in other plant species such as Dianthus...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
[2H5]indole-3-acetic acid	Olchemlm Ltd	031 1531	Internal standard for LC-MS/MS
[2H5]trans-zeatin	Olchemlm Ltd	030 0301	Internal standard for LC-MS/MS
[2H5]trans-zeatin riboside	Olchemlm Ltd	030 0311	Internal standard for LC-MS/MS
[2H6]N6-isopentenyl adenine	Olchemlm Ltd	030 0161	Internal standard for LC-MS/MS
[2H6]N6-isopentenyl adenosine	Olchemlm Ltd	030 0171	Internal standard for LC-MS/MS
indole-3-acetic acid	Wako	098 00181	standard for LC-MS/MS
trans-zeatin	SIGMA-ALDRICH	Z0876 5MG	standard for LC-MS/MS
trans-zeatin riboside	Wako	262 01081	standard for LC-MS/MS
N6-isopentenyl adenine	SIGMA-ALDRICH	D7674 1G	standard for LC-MS/MS
N6-isopentenyl adenosine	ACROS ORGANICS	22648 1000	standard for LC-MS/MS
acetonitrile hypergrade for LC-MS LiChrosolv	MERCK	1.00029.1000	solvent for LC-MS/MS
Water for chromatography LiChrosolv	MERCK	1.15333.1000	solvent for LC-MS/MS
HPLC	SHIMADZU	Prominence
MS	Sciex	3200QTRAP
Oasis HLB 30 mg/1 cc	Waters	WAT094225	cartridge column
Oasis MCX 30 mg/1 cc	Waters	186000252	cartridge column
screw neck total recovery vial	Waters	186002805
blue, 12 x 32mm screw neck cap and PTFE/silicone septum	Waters	186000274
Acquity UPLC BEH C18, 2.1x100 mm	Waters	186002350	UPLC column
Proshell 120 EC-C18, 2.1x50 mm	Agilent	699775-902	UPLC column
Digital microscope	Leica	DHS1000
TissueLyser II	QIAGEN	85300
Surgical blade	Feather	No. 22
Scalpel handle	Feather	No. 4
Savant SpeedVac/Refregerated vapor trap	Thermo Fisher Scientific	SPD111/RVT4104	vacuum concentrartor
Disposable glass tobe (13x100 mm)	IWAKI	9832-1310
Sterile petri dish	INA OPTICA	I-90-20