Materials and Reagents

Phytohormone standards and internal standards (Table 1).

Table 1. Phytohormone standards and internal standards.

Reagent	Supplier	Classification	Catalog number	Storage temperature (°C)
tZEATIN	OlChemim	STD	001 0301	-20
DHZ	OlChemim	STD	001 0601	-20
tZR	OlChemim	STD	001 0311	-20
DHZR	OlChemim	STD	001 0611	-20
tZMP	OlChemim	STD	001 5141	-20
iP	OlChemim	STD	001 0161	-20
iPR	OlChemim	STD	001 0171	-20
iPAMP	OlChemim	STD	001 5041	-20
IAA	OlChemim	STD	003 1531	-20
GA₁	OlChemim	STD	012 2491	-20
ABA	OlChemim	STD	013 2701	-20
D₅-tZ	OlChemim	ISTD	030 0301	-20
D₃-DZ	OlChemim	ISTD	030 0601	-20
d₅-tZR	OlChemim	ISTD	030 0311	-20
d₃-DZR	OlChemim	ISTD	030 0611	-20
d₅-tZRP	OlChemim	ISTD	030 0311	-20
d₆-iP	OlChemim	ISTD	030 0161	-20
d₆-iPR	OlChemim	ISTD	030 0171	-20
d₆-iPAMP	OlChemim	ISTD	030 5041	-20
d₅-IAA	OlChemim	ISTD	031 1531	-20
d₂-GA₁*	OlChemim	ISTD	032 2491	-20
d₂-GA₂₀	OlChemim	ISTD	032 2481	-20
d₂-GA₂₉	OlChemim	ISTD	032 2471	-20
d₆-ABA	OlChemim	ISTD	034 2721	-20

Tzeatin: trans-zeatin; DHZ: dihydrozeatin; tZR: trans-zeatin riboside; DHZR: dihydrozeatin ribodide; tZMP: trans-zeatin riboside-5'-monophosphate; iP: isopentenyladenine; iPR: isopentenyladenosine; iPAMP: isopentenyladenosine-5'-monophosphate; IAA: indole-3-acetic acid; ABA: abscisic acid; GA₁, ₂₀, and ₂₉: gibberellin A₁, A₂₀, and A₂₉; STD: phytohormone standard; ISTD: phytohormone internal standard.

*Can be replaced with d₄-GA₁ (product number: 032 2491) due to d₂-GA₁not being commercially available anymore.

Acetonitrile (Merck, catalog number: 1.00030)
Methanol (Merck, catalog number: 1.06007)
Milli-Q water (Merk Milli-Q)
Acetic acid (Merck, catalog number: 5.33001)
Formic acid (Merck, catalog number: 5.33002)
Liquid nitrogen
Node 2 axillary buds from garden pea plants with five fully expanded leaves
Internal standard working solution (see Recipes)
Extraction solvent (see Recipes)
1% acetic acid (see Recipes)
Plant hormone standard solutions (see Recipes)

Equipment

2010 Geno Grinder (SPEX SamplePrep, model: 2010)
3 mm diameter 440C stainless steel balls for Geno/Grinder 2010 (SPEX SamplePrep, product number: 2151)
Refrigerated centrifuge (Eppendorf, model: 5425R)
Solid phase extraction manifold (Chromabond, model: 730150)
Rotational vacuum concentrator with cold trap (Christ, model: RVC 2-33)
Ultra-high pressure liquid chromatograph system (Shimadzu Corporation, model: Nexera X2)
Triple quadrupole linear ion trap mass spectrometry system (AB Sciex, model: 5500)
Laboratory scale (accuracy: 0.0001 g)
Sep-Pak tC18 cartridge (Waters, catalog number: WAT036820)
1.5 mL Eppendorf tube (Eppendorf, catalog number: 0030121872)
HPLC vial (Agilent, catalog number: 5188-6591)
HPLC cap (Agilent, catalog number: 5190-7024)
Kinetex C18 reversed phase UPLC column (2.1 mm ×100 mm, 1.7 μm) (Phenomenex, catalog number: 00A-4475-AN)
Vertex low-retention non-filtered pipette tip (SSIbio, catalog number: 4337N00)
15 mL Falcon tube (Corning, catalog number: 352096)
4 °C refrigerator and -20 °C and -80 °C freezer

Procedure

Homogenization and extraction of samples

Weigh a 1.5 mL Eppendorf tube with two grinding beads (3 mm stainless steel metal balls) inside.
Sample 20 pea axillary buds (approximately 2 mm each) into one Eppendorf tube and snap freeze in liquid nitrogen.
Weigh the Eppendorf tube again after sampling and calculate the weight of axillary buds.
Homogenize the frozen plant tissues with 2010 Geno Grinder at 4 °C (1,500 stroke/min, 2×1 min).

Two outputs: RNA pellet and supernatant

Add 1 mL of freshly prepared extraction solvent (80% acetonitrile containing 1% acetic acid and 5 µL internal standard working solution) to the homogenized sample and vortex for 10 s at room temperature.
Leave the extract at -20 °C for 5 min and then centrifuge at 15,900 × g and 4 °C for 10 min.
Transfer 950 μL of supernatant to a new Eppendorf tube for hormone extraction. The remaining pellet can be used for RNA extraction (see Note 1).
Evaporate supernatant using a rotational vacuum concentrator with cold trap at room temperature until pellet is completely dry.

Sample cleaning with solid phase extraction (SPE) column

Add 1 mL of 1% acetic acid to the dried sample and pipette up and down three times for redissolution.
Store the sample at 4 °C until loading on Sep-Pak tC18 cartridge at step 14.
Set up the solid phase extraction manifold with Sep-Pak tC18 cartridge (Figure 1).

Figure 1. The setup of solid phase extraction manifold with Sep-Pak tC18 cartridge.
Wash Sep-Pak tC18 cartridge with 1 mL of 100% methanol.
Activate tC18 cartridge with 1 mL of 1% acetic acid.
Load the sample (from step 10) on the activated cartridge.
Wash the loaded cartridge with 1 mL of 1% acetic acid.
Elute the sample with 1 mL of 80% acetonitrile containing 1% acetic acid.
Evaporate the eluted sample using the rotational vacuum concentrator with cold trap at room temperature until completely dry.
Add 50 µL of 1% acetic acid to the dried sample, pipette up and down three times for redissolution, and vortex for 30 s.
Centrifuge the dissolved sample at 15,900 × g and 4 °C for 10 min and transfer the supernatant into a HPLC vial.
Store the sample at -80 °C before loading on the UPLC–MS/MS.

UPLC–MS/MS setup

The UPLC–MS/MS system used for this protocol is a Nexera X2 ultra-high pressure liquid chromatograph system coupled with a 5500 triple quadrupole linear ion trap mass spectrometry system equipped with an electrospray ionization source (ESI). The UPLC method is as follows:

Mobile phase A: 0.5% formic acid in Milli-Q water (v/v).

Mobile phase B: 0.5% formic acid in acetonitrile (v/v).

Flow rate: 0.5 mL min^-1.

UPLC gradient: 4% B over 0.5 min, 4%–15% B over 7 min, and 15%–95% B over 3.5 min.

Cleanup step: 95%–95% B over 2 min, 95%–4% B over 0.1 min, and column wash for 1 min.

ESI parameters (for positive and negative modes, respectively): curtain gas, 20 psi; collision gas, medium; ion source temperature, 500 °C; ion source gas 1 and 2, 80 psi; and IonSpary voltage, +4,500 V or -4,500 V.

The scheduled multiple reaction monitoring (sMRM) parameters are listed in Table 2.

Table 2. sMRM parameters for phytohormone standards and their corresponding internal standards. CE, DP, and EP are the same between PH and ISTD.

PH	Q1	Q3	RT	SM	ISTD	Q1	Q3	RT	SM	CE	DP	EP
tZEATIN	220	136	2.3	+	d5-tZ	225	136	2.2	+	25	75	10
DHZ	222	136	2.5	+	d3-DZ	225	136	2.4	+	25	100	8
tZR	352	220	3.5	+	d5-tZR	357	225	3.3	+	25	80	10
DHZR	354	222	3.6	+	d3-DZR	357	225	3.4	+	30	80	10
tZMP	432	220	1.9	+	d5-tZRP	437	225	1.8	+	25	100	10
IP	204	136	5.4	+	d6-IP	210	136	5	+	20	70	10
IPR	336	136	6.9	+	d6-IPR	342	136	6.5	+	40	80	10
IPAMP	416	136	4	+	d6-IPRP	422	136	3.5	+	42	100	10
IAA	176	130	8.1	+	d5-IAA	181	135	8.1	+	25	120	15
GA1	347	229	7.6	-	d2-GA1	349	231	7.6	-	-40	-80	-15
GA20	331	287	9.5	-	d2-GA20	333	289	9.5	-	-30	-80	-15
GA29	347	303	4.8	-	d2-GA29	349	305	4.8	-	-30	-80	-15
ABA	263	153	9.4	-	d6-ABA	269	159	9.4	-	-20	-80	-15

PH: phytohormone; Q1: precursor ion selected in Q1; Q3: product ion selected in Q3; RT: retention time; ISTD: internal standard; SM: scan mode; CE: collision energy; DP: declustering potential; EP: entrance potential.

Data analysis

MultiQuant software (AB Sciex, USA) is used to analyze raw mass spectrometry data. The concentration of each hormone is further calculated using Microsoft Excel by comparing with the internal standard concentration added in the sample.

Notes

The RNA from the plant debris pellet should be able to be extracted using most available RNA extraction methods. Two established RNA extraction methods have been tested: the first is a commercial RNA extraction kit, and the second is a cetyltrimethylammonium bromide (CTAB)-based method (Barbier et al., 2019a; Cao et al., 2020). The RNA from the pellet is extracted immediately, and the preservation condition for the pellet has not been tested in this protocol.
The validation of the UPLC–MS/MS method has been detailed in our previous publication (Cao et al., 2020).
Plant material should always be kept frozen until adding the extraction solvent, to prevent degradation of plant hormones or possible metabolic reactions in the plant cell.
Sep-Pak tC18 cartridge should be kept wet after activation until the elution of the sample.
The maintenance of the UPLC–MS/MS system should be strictly followed as per the requirement of the manufacturer, to prevent sensitivity decrease and environmental contamination.
All solvents and consumables that directly contact the sample have been tested and are free of environmental contamination. If any consumables need to be replaced with different brands, environmental contamination needs to be tested.
If the UPLC–MS/MS system needs to be replaced with other types of models or brands (e.g., Agilent, Thermos, or Waters), the settings of the UPLC–MS/MS need to be reoptimized with plant hormone standards and internal standards.
If the plant hormone extraction process cannot be finished in one working day, the process can be paused after step 8, and the dried sample can be stored at -20 °C for 24 h before continuing the extraction.
A summarized flow chart for the phytohormone and RNA extraction can be found at https://www.frontiersin.org/files/Articles/605069/fpls-11-605069-HTML/image_m/fpls-11-605069-g001.jpg.

Recipes

Internal standard working solution
10 ng mL^-1 d₅-tZEATIN
20 ng mL^-1 d₃-DHZ
40 ng mL^-1 d₅-tZR
100 ng mL^-1 d₃-DHZR
120 ng mL^-1 d₅-tZMP
40 ng mL^-1 d₆-iP
60 ng mL^-1 d₆-iPA
10 ng mL^-1 d₆-iPRMP
200 ng mL^-1 d₅-IAA
200 ng mL^-1 d₂-GA₂₀
200 ng mL^-1 d₂-GA₂₉
200 ng mL^-1 d₆-ABA
Dissolved in 100% methanol.
Store at -80 °C for up to one month.
Extraction solvent
Dilute acetonitrile and acetic acid with Milli-Q water, to 80% acetonitrile containing 1% acetic acid. Store at 4 °C for up to one month.
1% acetic acid
Dilute acetic acid with Milli-Q water to 1% acetic acid. Store at 4 °C for up to one month.
Plant hormone standard solutions
Plant hormone standard and internal standard solutions were diluted with methanol to 100 µg mL^-1 (stock solution) and 1 µg mL^-1 (working solution). Store at -80 °C for up to one year.

Acknowledgments

This method has been published as a methodology article in Frontiers of Plant Science (Cao et al., 2020). This research was funded by The University of Queensland and the Australian Research Council Georgina Sweet Fellowship (FL180100139) and supported during the establishment phase of the ARC Centre of Excellence for Plant Success in Nature and Agriculture (CE200100015). We thank Dr. Lindsay Shaw for reviewing the manuscript.

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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