Plant Growth Regulators

To determine levels of GA in anthers and filaments, flowers at stage 13 (ref. 54 (link)) were dissected using a stereomicroscope. The GA levels in whole flowers at approximately stage 13 were also analysed. To measure the level of hormones in seedlings, 8-day-old plants grown on vertical plates were separated into shoots and roots. To collect developing seeds, siliques at 10 DAF were dissected using a stereomicroscope. All samples were frozen in liquid nitrogen and weighed after lyophilisation.
The samples were ground and homogenized in extract solution (Supplementary Table 2) with defined amounts of deuterium-labelled internal standards. The mixtures were incubated for 12 h at 4 °C and then centrifuged at 3,000g for 20 min at 4 °C. The supernatants were dried in a vacuum and dissolved in 1 ml of water containing 1% (v/v) acetic acid. After several steps of purification on solid phase columns, extracts were dried in a vacuum and dissolved in 20 μl of water containing 1% (v/v) acetic acid. The purification steps are summarized in Supplementary Tables 2 and 3. The LC–MS/MS system consisting of a quadrupole/time-of-flight tandem mass spectrometer (Triple TOF 5600, AB SCIEX), and a Nexera HPLC system (SHIMADZU) were used in these analyses. LC separations were performed at a flow rate of 400 μl min⁻¹ using the conditions presented in Supplementary Table 4. MS/MS conditions are presented in Supplementary Table 5. We used a software tool (MultiQuant 2.0, AB SCIEX) to calculate plant hormone concentrations from the LC–MS–MS data.

For external calibration and instrument detection (LOD_i) and quantification (LOQ_i) limit determination, the mixture containing 1 mmol/L of each individual standard was serially diluted with 20% aq. methanol by 2.0 – 2.5-fold increment to obtain 23 concentration steps (0.01 nmol/L – 500 μmol/L). The method limits of quantification (LOQ_m) were determined by the standard addition method [37 ]. For this, 20 mg of lyophilized plant pool (i.e. a mixture consisting of tomato leaves and roots, potato leaves, rice leaves and roots, barley leaves and Arabidopsis leaves mixed in equal proportion) was spiked with a standard mixture containing ²H₆-SA, ²H₆-ABA, ²H₆-JA, ²H₂-(−)-JA-Ile, ²H₅-OPDA at eight concentration levels (0.14 – 27.33 μg/L). The spiked samples were extracted with methanol and subjected to SPE as described above.
Recoveries of phytohormones on PS/DVB-based Cromabond® HR-XC material (Macherey & Nagel, Düren, Germany) were determined by the standard addition method. SA, ABA, JA, 12-OH-JA, JA-Ile and OPDA were diluted in 500 μL aliquots of methanol at the concentrations of 0, 1.3, 2.5, 5.0, 10.0 and 15 μg/L and processed with and without extraction of complex lyophilized plant pool (20 mg). The obtained extracts were subjected to RP-LC-MS/MS with and without further drying in nitrogen stream. The residues were reconstituted in 40 μL acetonitrile before further dilution to 80 μl with 0.3 mmol/L aq. ammonium formate (pH 3.5).
The analyte abundances expressed as peak heights were plotted against their concentrations. Additionally, regression coefficients and R² values were calculated.

Liquid chromatography was carried out using a UFLC with an autosampler (Shimadzu Corporation, Kyoto, Japan). A Waters Atlantis T3 (Waters Corporation) column (2.1 × 150 cm, 3 μm) was used at ambient temperature. The injected volume of sample was 10 μL. The elution gradient was carried out with binary solvent system consisting of 0.02% acetic acid in H₂O (solvent A) and 0.02% acetic acid in MeCN (solvent B) at a constant flow rate of 250 μL/min. A linear gradient profile with the following proportions (v/v) of solvent B was applied: gradient profile 0 to 5 min and 0% of B, 5 to 8 min and 0% to 16% of B, 8 to 20 min and 16% to 100% of B, 20 to 25 min and 100% of B, 25 to 28 min and 100% to 0% of B, and 28 to 32 min with 4 min for re-equilibration and 0% of B.
To diagnose the hormone precursor-to-product ion transitions, mixtures of 150 ng/mL of the standard compounds dissolved in 50% MeCN were directly infused into a hybrid triple quadrupole/linear ion trap mass spectrometer (ABI 4000 Q-Trap, Applied Biosystems, Foster City, CA, USA) outfitted with an electrospray ion source. The analysis parameters were optimized for the production of characteristic precursor-to-product ion transitions in negative or positive ionization modes. ABA, IAA, IAA-Asp, JA, JA-Ile, SA, sakuranetin, naringenin, and their internal standards were scanned in the negative mode, whereas momilactone A and MeJA were analyzed in the positive mode. The mixtures of standard compounds were separated by reversed-phase UFLC and analyzed by ESI-MS/MS in the MRM mode with 50 ms dwell time, 5 ms of pause time between mass ranges, and 700 ms of settle time for switching polarities. The identities of phytohormones and metabolites in the crude plant extracts were confirmed by analysis of product ion fragments obtained by the hybrid triple quadrupole/linear ion trap mass spectrometer, operating in the IDA mode, with a source voltage of 4.5 kV and source temperature of 550. In the ''Enhanced Product Ion" scan mode, precursor ions were fragmented with collision energy +25 kV or -25 kV and products in the range of 50 to 500 m/z were detected.
UFLC-ESI-MS/MS assays were repeated twice biologically, with each repetition having three replicates. Similar results were obtained in repeated experiments; only the result in one repetition was presented.

To assess general statistical importance of hormone treatments in the metabolite profiles, a Principal Component Analysis (PCA) was performed on the normalized data (Fig. 2). The hormone type appeared to be responsible for the variation observed in PC1 (which explained 17.7%) while variation between the specific phytohormones and the treatment concentrations appeared to be responsible for PC2 (9.18% explained variance). Overall, a clear separation between growth hormones (pink-purple) and stress-response hormones (blue-green) was observed. A PCA of the full feature table was also conducted and a similar separation was observed (Supplementary Information).
Fig. 2

PCA of the normalized feature table. Circles were added manually with growth treatments (coloured pink-purple) circled in pink and stress-response treatments (coloured blue-green) circled in blue. As the broken stick test revealed that the PC1 and PC3 axes explained more variance than would be expected by randomly dividing the variance into parts, PC1 and PC3 were chosen for the plot. Results of the broken stick test are available in the Supplement and Zenodo

Analysis of the chemodiversity showed unique patterns with respect to the features detected in each treatment (Fig. 3). The number of features detected was consistent between all the treatments except for NAA10 which had significantly fewer features (Fig. 3a). The Pielou’s evenness (J) for all treatments was consistent, with the exception of SA1 and NAA10 which had significantly higher and lower J values, respectively (Fig. 3b). No significant differences were observed in the Shannon diversity (H’) (Fig. 3c). Determining the Functional Hill diversity, which also does take the dissimilarity of features into account (Petrén et al., 2022 ), showed that the significantly lower diversity in NAA10 was explained by structurally similar features with less abundance than the other treatments and a slightly higher dissimilarity of features in BAP10, G, and S (Fig. 3d).
Fig. 3

Diversity indices of the features detected in the MS1 data. A: number of features, B: Pielou’s evenness (J), C: Shannon diversity Index (H’). D: Functional Hill diversity. S: stress-response control, G: growth control

The salt-tolerant XuShu 22 seedlings were grown in 1/4 Hoagland solutions and used for all the stress and hormone treatments with three different biological replicates. The roots were immersed in 150 mM NaCl and 20% PEG6000 solutions, respectively, for salt and dehydration stress treatment, then root samples were collected. Seedlings were incubated at 4°C and 42°C, respectively, for cold and heat conditions, then leaves samples were harvested. And the leaves of seedlings were collected after spraying with 0.1 mM ABA or 2 mM SA solutions for phytohormone tests (Meng et al., 2018 (link)). All of the samples were collected at 0, 1, 12, 24, and 48 hours after each treatment.
Total RNA was extracted using RNA Extraction Kits (TianGen, Beijing, China) following the manufacturer’s instructions. PrimeScript reverse transcriptase with the gDNA Eraser (TaKaRa, Dalian, China) was used to reverse-transcribe 1 μg of each RNA sample. qRT-PCR tests were conducted using the CFX96TM Real-Time System as previously described (Bio-Rad, USA) (Meng et al., 2020a (link)), and the sweetpotato ARF gene (JX177359) was employed as an internal control (Park et al., 2012 (link)). All the qRT-PCR primers are provided in Supplementary Table S4.

The promoter regions of the BnGR2R3-MYB genes, 1,000 bp genomic DNA upstream sequences of each of the 105 BnGR2R3-MYB were selected, and the cis-elements predicted using PlantCare (http://bioinformatics.psb.ugent.be/web tools/plantcare/html/). Response class elements such as light-responsive, plant growth, stress-responsive and phytohormone-responsive were filtered and the cis-acting elements visualized in TBtools, and displayed with a heatmap. Following blasting in BLASTP against Swissport database, functional annotation of BnGMYB proteins was performed in Blast2GO Tool and subsequent mapping Gene Ontology (GO) terms, and visualization with R ggpolt2.