Transcriptomic and hormonal dynamics in relation to adventitious rooting of two parental Petunia species highlight a coordinated activation of the jasmonate and auxin pathways and an important role of upper-shoot-derived auxin influx

Introduction: Adventitious rooting of cuttings is a key developmental process for the vegetative propagation of many crops that involves phytohormone-controlled reprogramming and redifferentiation of specific cells in the stem base. The endogenous control of phytohormone action at the whole-plant level is not completely understood.

Methods: Using the genome-sequenced Petunia axillaris and Petunia inflata, we monitored the transcriptome of phytohormone-related genes and phytohormone levels in different cutting sections through a phytohormone-targeted microarray, RT-qPCR, and LC-MS/MS, and analyzed the rooting response to manipulations of auxin levels and transport.

Results: In the stem base of both species, genes controlling jasmonic acid (JA) biosynthesis, conjugation, and signaling, and encoding transcription factors of the ERF family were already upregulated at 0.5 hours post excision (hpe), followed by increased regulation of auxin-related genes. Accordingly, JA and its physiologically active isoleucine conjugate JA-Ile accumulated transiently at 0.5 hpe, before indole-3-acetic acid (IAA) peaked at 2 hpe. Genes controlling auxin biosynthesis were mostly downregulated, whereas three IAA-leucine-resistant-like genes were strongly upregulated between 0.5 and 2 hpe. P. inflata’s greater rooting capacity compared with P. axillaris was linked to higher stem-base IAA levels (0–72 hpe), resulting in a higher IAA/cytokinin ratio and stronger upregulation of auxin-signaling genes. P. inflata showed a steeper IAA gradient between the leaves and the stem base, which was positively and negatively correlated with leaf salicylic acid and cytokinin isopentenyladenine levels, respectively, and associated with exclusive upregulation of PIN-like genes in the leaves. P. axillaris showed a stronger improvement in rooting with low IAA doses than P. inflata. Blocking polar auxin transport in the upper shoot prevented rooting in both species.

Discussion: The results reveal excision-triggered coordination of jasmonate and auxin pathways in the stem base, interacting with ERF transcription factors, and indicate an important role for upper shoot-derived auxin influx, potentially regulated by salicylic acid and cytokinins. Higher rooting capacity of P. inflata can be explained by the higher IAA level in the stem base. The results indicate important roles of ERF113/114, ILR-like2 and 6, PIN6, PIN-like 1/3, the PINOID gene A4A49_10797, ARF11, and several LBD genes in adventitious rooting of Petunia.

Introduction

Adventitious root (AR) formation in cuttings is a key developmental process for the vegetative propagation of many horticultural and forestry crops that requires a reprogramming of particular responsive cells in the stem base near the wound and is evoked by two stimulating principles: wounding and isolation from the donor plant (reviewed in Druege et al., 2016 and Druege et al., 2019). It involves sequential phases. According to Da Costa et al. (2013) and Druege et al. (2019), three phases are distinguished. The first, the so-called induction phase, is characterized by an anatomical lag phase devoid of cellular changes, during which the initial cell reprogramming occurs. After the determination of AR founder cells, the initiation phase starts with qualitative changes in cell structures, followed by cell division and differentiation of the new cell clusters into dome-shaped root primordia. The final expression phase begins with the differentiation of primordia into the complete root body, with differentiated vascular bundles connected to the vascular cylinder of the stem, followed by the emergence of roots.

Research over several decades has shown that the rooting success of cuttings is dependent on primary metabolic processes but particularly on the action of plant hormones, while both factors are interrelated (reviewed in Da Costa et al., 2013; Druege et al., 2019; Lakehal and Bellini, 2019). Among the plant hormones, auxin plays an outstanding role. It acts as a positive regulator during the induction phase, whereas high auxin levels have an inhibitory role during subsequent differentiation and growth of ARs (Da Costa et al., 2013). Even though synthetic auxins have frequently been used to stimulate the rooting of cuttings, the major endogenous bottlenecks of auxin action at the whole-cutting level and the interactions with other plant hormones are not completely understood. Cytokinins act antagonistically against auxin during AR induction but, at low concentrations, have important functions during early cell reprogramming (reviewed in Da Costa et al., 2013). Some studies point to positive effects of ethylene during the early induction of ARs, possibly contributing to redifferentiation, while it also can interact with auxin in both directions (reviewed in Druege et al., 2019). Other studies suggest a positive regulator function of jasmonic acid (JA) during AR induction in cuttings (reviewed in Druege et al., 2019), which, however, stands in contrast to an inhibitory function of JA during etiolation-induced AR initiation in hypocotyls of intact Arabidopsis seedlings (Gutierrez et al., 2012).

Mostly, leafy shoot tip cuttings are used for vegetative propagation, which are not formed by the model plant Arabidopsis. Among horticultural plants, Petunia hybrida has an economically important role worldwide, and many cultivars are propagated by leafy shoot tip cuttings. Petunia has been established as a model for molecular investigations of diverse research questions (Gerats and Vandenbussche, 2005; Vandenbussche et al., 2016). We have previously used the model cultivar P. hybrida Mitchell and characterized the endogenous regulation of excision-induced AR formation. AR formation involves early accumulation of JA (Ahkami et al., 2009) and of indole-3-acetic acid (IAA) (Ahkami et al., 2013) in the stem base during the root induction phase. The accumulation of IAA and AR formation is dependent on a functioning polar auxin transport (PAT) in the cuttings, and PAT-controlled IAA accumulation is important not only for AR induction but also for the activation of invertases that control the establishment of the new sink in the stem base (Ahkami et al., 2013). Excision-induced AR formation of P. hybrida Mitchell involves differential expression of genes putatively controlling ethylene (ET), strigolactone (SL), and auxin homeostasis and signaling in the stem base, while rooting is dependent on ethylene biosynthesis and perception (Druege et al., 2014; Bombarely et al., 2016). The transcriptome data of these studies were limited as based on available ESTs and had not been linked to hormone data. In addition, the contribution of the upper cutting parts to excision-induced AR formation in the stem base of Petunia is still unknown.

Recently, the complete genomes of Petunia axillaris and Petunia inflata, which constitute important parental species of modern P. hybrida cultivars, have been sequenced (Bombarely et al., 2016). Preliminary studies indicated that both P. axillaris and P. inflata are easy to root, while P. inflata showed more intense rooting than P. axillaris.

In light of these findings and the still open questions, the present study aimed to address the following:

● Simultaneous analysis of the excision-induced dynamics of the phytohormone-related transcriptome and the phytohormone concentrations in the stem base and upper cutting sections of P. axillaris and P. inflata.

● Identification of important hormonal events and candidate genes that putatively control AR formation in both species and may contribute to the difference in rooting efficiency.

A phytohormone-targeted microarray, reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR), and LC-MS/MS were combined with local pharmacological manipulations of auxin levels and transport to elucidate these relationships.

Materials and methods

Plant material, growth conditions, and analysis of rooting

Donor plants of P. axillaris N and P. inflata S6 were established from seeds and cultivated in the greenhouse as previously described for P. hybrida Mitchell (Klopotek et al., 2010). After the donor plants reached 3 months of age, shoot tip cuttings, as illustrated in Figure 1, were excised at regular intervals over a period of up to 6 months and used for the experiments, always leaving two leaves or nodes on the stock plant. Cuttings were planted in perlite Perligran A (Knauf Perlite GmbH, Dortmund, Germany) and cultivated in a growth chamber under the following conditions: temperature, 22°C/20°C (day/night); humidity outside the covered trays, 85%/60% (day/night); PPFD of 100 μmol m⁻² s⁻¹ at plant level during a 10-h photoperiod provided by white fluorescent tubes. During the rooting period, cuttings were manually watered and did not receive nutrients or phytohormones unless indicated otherwise (Figure 1). At specified days post excision (dpe) of the cuttings (Figure 1), ARs were counted and assigned to different root length classes (in 1-cm increments). The number of ARs formed per planted cutting, the mean length per AR, and the total root length per planted cutting were calculated as described by Druege et al. (2007) and Agulló-Antón et al. (2011).

Figure 1

Figure 1. Experimental setup of transcriptome, phytohormone, and rooting analysis of cuttings of P. axillaris and P. inflata, and of IAA and NPA applications. SA, shoot apex including the smallest adjacent leaves that were maximally 1 cm in length; UL, upper leaves; BL, basal leaves; SB, stem base, 0.5 cm in length; US, upper stem, the stem section between SB and SA; hpe, hours post excision; dpe, days post excision.

Sampling and grinding

At specified hours post excision (hpe) of the cuttings, according to Figure 1, their stem bases (SB, 0.5 cm in length), upper stems (US), longitudinal halves of the two basal leaves (BL) and the remaining upper leaves (UL), and shoot apices including the smallest adjacent leaves (SA) were shock-frozen in liquid nitrogen and stored at − 80°C. For each replicate (n = 3), pooled material from 10 or 16 cuttings was ground manually in liquid nitrogen using a mortar and pestle, and aliquots of 100 and 50 mg fresh mass were used for extraction and analysis of the transcriptome and the phytohormones, respectively.

RNA extraction

Total RNA was extracted from the Petunia cutting samples following the protocol of the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Up to 100 mg fresh mass was processed, according to the lysis capacity of the RLT buffer and RNeasy spin columns. Following the manufacturer’s protocol, 30 µL of total RNA was obtained. RNA integrity was checked on an agarose gel, and total RNA concentration and quality were determined using a NanoDrop™ 2000/2000c spectrophotometer (Thermo Fisher Scientific, Schwerte, Germany). Only high-quality RNA samples (OD260/280 = 1.8 − 2.2, OD260/230 ≥ 2) were used. The integrity of RNA used for microarray analysis was additionally verified using an Agilent 2100 Bioanalyzer (Agilent Technologies Deutschland GmbH, Waldbronn, Germany).

Design and use of the phytohormone-targeted microarray

Based on previous findings on the hormonal regulation during AR formation in P. hybrida Mitchell, a phytohormone-targeted microarray was developed that covered those genes from P. axillaris and P. inflata that putatively control homeostasis, signal transduction, and downstream responses of/to auxin, JA, and SLs. To also cover the response of AR formation to ET, genes encoding ethylene response factors (ERFs) that may respond to ET and other phytohormones such as JA (Heyman et al., 2018) were included as the main gene family of the gene category “hormonal interaction” (short name “interaction”). To compile the gene list for the microarray, literature research was first carried out, and genes or gene families involved in the pathways mentioned above were selected. Subsequently, the Petunia axillaris v1.6.2 CDS and Petunia inflata v1.0.1 CDS databases (solgenomics.net) were searched for the respective annotated genes. Furthermore, the NCBI database was searched for homologous genes in other Solanaceae species, and the corresponding sequences (CDS or AAS) were blasted against the Petunia genomes. The results of the two BLAST searches were compared, and the gene list was adjusted accordingly. Later, based on the results of microarray, annotation of differentially expressed genes of the families YUCCA family of flavin monooxygenase (YUCCA), IAA-leucine resistant (ILR), ILR-like (ILL), pin-formed (PIN), PIN-like, Auxin indole-3-acetic acid protein (Aux/IAA), auxin response factor (ARF), lateral organ boundaries domain (LBD), PROTEIN serine/threonine kinase pinoid (PINOID), and ERF was updated using a new version of the P. axillaris genome 4.02 I, with the kind permission of Cris Kuhlemeier, University of Bern, Switzerland (later published as Pax403 under https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_026929995.1/).

Based on the genome sequences of 1,265 genes of interest (605 from P. axillaris and 660 from P. inflata), an Array-to-Go Custom microarray was designed by OakLabs GmbH (Henningsdorf, Germany). For this purpose, five to 10 specific isothermal probes, each 45–60 bp in length, were designed for each gene of interest. An additional 1,000 probes from a previously used microarray (Yang et al., 2019) were used for normalization. The probes were spotted on a microarray. Extracted RNA samples were labeled with a Cy3 fluorescent dye and hybridized to the microarray, which was manufactured in collaboration with Agilent (Santa Clara, CA, USA). The best-performing oligonucleotide probe(s) were selected for each gene based on the hybridization data and used for the subsequent data analysis. Quantile-normalized expression values of the genes of interest were provided and then analyzed as described below.

cDNA synthesis and reverse transcription real-time quantitative PCR analysis

Transcription of selected genes was additionally analyzed by RT-qPCR analysis. First-strand complementary DNA (cDNA) was synthesized from RNA using the PrimeScript RT Master Mix reverse transcriptase (Takara Bio Europe SAS, Saint-Germain-en-Laye, France) according to the manufacturer’s protocol. Four microliters of diluted RNA (125 ng µL⁻¹) were added to 1 µL of 5× PrimeScript RT Master Mix. Primers, which are described in Supplementary Table S1, were selected using Primer3web version 4.1.0 software and obtained salt-free at 0.01 µM from Eurofins (Jena, Germany). RT-qPCR was performed in plates (Brand GmbH + Co KG, Wertheim) using iQ SYBR Green Supermix (Bio-Rad Laboratories GmbH, Feldkirchen, Germany) in a 10-µL reaction volume with three technical replicates per biological sample. The reaction contained 2.5 µL of primer mix, with 1/100 (Fw + Rv) diluted primers, 5 µL of SYBR Green, and 2.5 µL of diluted cDNA (1:20). Melting curve analysis confirmed the specificity and quality of the PCR products. RT-qPCR was conducted on the CFX96 Real-Time System (Bio-Rad) with an initial denaturation for 3 min at 95°C, followed by 40 cycles of 10 s at 95°C and 40 s at 60°C. After preliminary tests according to Mallona et al. (2010), ribosomal protein S13 (Ph-RPS13) and elongation factor 1-alpha (EF1α) were used as reference genes. Cq values were calculated using the Bio-Rad CFX Maestro software. Relative transcript levels were determined by the 2^−ΔΔCT method (Livak and Schmittgen, 2001) with geometric averaging of the reference genes (Vandesompele et al., 2002).

Plant hormone analyses

Hormone extraction

Fifty milligrams (fresh weight) of homogenate were extracted using three rounds with 80% methanol acidified to pH 2.4 with hydrochloric acid. For round 1, 400 µL; for round 2, 200 µL; and for round 3, 100 µL of solvent were used. Cell extraction was performed in 2 mL cryotubes with reinforced walls (710768, Biozym Scientific GmbH, Hessisch Oldendorf, Germany). To enhance cell rupture, one steel bead of 3 mm diameter, three steel beads of 1 mm diameter, and 200 mg glass beads with a diameter of 0.75–1 mm (Carl Roth GmbH, Karlsruhe Germany) were added to each tube, and bead milling was performed for 3 × 1 min in a homogenizer (FastPrep-24, MP Biomedicals, Eschwege, Germany). The combined extracts were pooled, centrifuged, and stored on ice until measurement on the same day.

Online SPE-UPLC-MS/MS phytohormone analysis

Hormones were analyzed by online solid-phase extraction coupled ultra-high-performance liquid chromatography–tandem mass spectrometry (SPE-UPLC–MS/MS) using a CTC Combi-PAL autosampler with a 1,000-µL injection loop, a 96-well rack of a CHROspe divinylbenzene polymer SPE phase (10 mm × 2 mm; particle size: 25–35 µm; fill weight, 10 mg), a SPH1299 UPLC gradient pump, an EPH30 UPLC dilution pump, a Mistral column oven (all Axel Semrau GmbH, Sprockhövel, Germany), and a mass spectrometer (QTrap 6500, ABSciex, Darmstadt, Germany). For each sample, 600 µL of plant extract was injected into the online SPE at a rate of 200 µL min⁻¹, where phytohormones were trapped by simultaneous addition of excess water (3,800 µL min⁻¹) before SPE. Sequential transfer of trapped hormones from the SPE cartridge to the UPLC column was accomplished by 120 µL of 20% acetonitrile and simultaneous water dilution prior to UPLC. For chromatographic separations by UPLC, a Nucleoshell RP Biphenyl column (100 mm × 2 mm × 2.7 µm, Macherey & Nagel, Düren, Germany) was used. The gradient was as follows: 0–2 min, 5% B; 2–13 min, linear gradient to 95% B; 13–15 min, 95% B; and 15–18 min, 5% B. The column temperature was 40°C, and the flow rate was 400 µL min⁻¹. Solvent A was 0.3 mM ammonium formate, acidified with formic acid to pH 3.0, and solvent B was acetonitrile. The autosampler temperature was maintained at 4°C. Mass-spectrometric detection of the phytohormones on a QTrap 6500 (ABSciex) was accomplished by electrospray ionization and multiple reaction monitoring (MRM) using the parameters in Supplementary Table S2. For this, the ion source was heated to 450°C. Curtain gas was set to 35 psi, ion source GS1 to 60 psi, and GS2 to 70 psi. The electrospray ionization voltage was 5,500 V in positive mode and − 4,500 V in negative mode. Fully automated sample processing was controlled by Chronos (Axel Semrau GmbH) via contact closure to trigger MS/MS runs controlled by Analyst 1.7.1., based on protocols described by Balcke et al. (2012) and Ai et al. (2023).

Pharmacological treatments

The rooting response of the two Petunia species to increasing external supply of IAA to the stem base during the root induction phase was analyzed under the same climate chamber conditions described above. For this purpose, cuttings, as described before, were immediately after excision placed in 25 mL vials (Figure 1A) containing one-half MS medium (Murashige and Skoog, 1962) supplemented with vitamins according to Gamborg et al. (1968) and adjusted to a pH of 5.8. The medium was supplemented with different concentrations of IAA (0, 5, 10, 50, and 150 mg L⁻¹). The vials were sealed with paraffin to prevent water evaporation, with a small cut large enough for the cutting to pass through so that the stem base was immersed in the medium (Figure 1A). The cuttings were kept in the vials until 72 hpe and then replanted in hormone-free perlite and cultivated until rooting was assessed.

To test the dependency of AR formation in the stem base on PAT-derived auxin influx from the upper shoot of the cutting, including all leaves, immediately after excision, rings of lanolin paste (Carl Roth GmbH) containing 0, 10, or 30 mg L⁻¹ 1-N-naphthylphthalamic acid (NPA; Duchefa, Haarlem, The Netherlands), were carefully applied around the petioles of the two basal leaves and the adjacent upper shoot, as illustrated in Figure 1B.

Data analysis

Mean values and standard error (SE) at the experimental level, or standard deviation (SD) over several replicative experiments, were calculated for the rooting data, the expression values of specific genes analyzed by microarray and RT-qPCR, and the phytohormone levels. The microarray data were analyzed using the CLC Genomics Workbench (Qiagen, Hilden, Germany). The normalized transcript levels determined at 0.5, 2, 24, and 72 hpe (x) were related to the respective levels at 0 hpe (y), the time of excision, to calculate fold changes (FC). These fold changes were log-transformed to log2 FC (= log2 [x/y]). If the log2 FC values were > 2 or < −2 and the p-values of expression values were < 0.05 (t-test), the fold changes were considered significant. Numbers of up- and downregulated genes (DEGs) of different categories were counted. Ratios of DEGs were calculated based on the total number of respective genes covered by the microarray. Heat maps were developed to illustrate the intensity of regulation for specific genes. Root number and length, RT-qPCR and microarray data from selected genes, and phytohormone levels were further analyzed using Statistica Version 3, TIBCO Software Inc. (Palo Alto, CA, USA). Depending on the number of factor levels, the significance of differences was analyzed by t-test or by ANOVA followed by Tukey test (p < 0.05). Applied statistical tests and the number of replications (n) are provided with the data. Pearson correlation coefficients and slopes of linear regressions were determined to analyze interrelationships between specific data using the “trend” function of Microsoft Excel (Munich, Germany).

Results

Rooting capacity of P. axillaris and P. inflata

Figure 2 summarizes the results of eight independent experiments analyzing the rooting capacity of P. axillaris and P. inflata. The early detection of ARs on cuttings of both species after a period of 16 to 18 days post excision demonstrates that both P. axillaris and P. inflata are easy to root. However, higher mean values of root number (Figure 2A), mean root length (Figure 2B), and total root length (Figure 2C) were observed for P. inflata across the eight experiments when compared with P. axillaris. P. inflata produced at least six roots and a total root length of at least 5 cm, whereas the minimum number and total length of roots for P. axillaris were 1 and 0.5 cm, respectively. These results indicate a higher rooting capacity for P. inflata than for P. axillaris.

Figure 2

Figure 2. Rooting capacity of P. axillaris and P. inflata. The number of roots per planted cutting (A), the mean length per root (B), and the total root length per planted cutting (C) were determined in eight independent experiments (Exp). Roots were analyzed on day 16 (Exp 1–2, 4–6), day 17 (Exp 3, 8), and day 18 (Exp 7). Columns and whiskers represent the mean values ± standard deviation calculated over all experiments. Individual dots show the mean values per experiment. The experiments involved three (Exp 4–7) or four (Exp 1–3, 8) replications, each consisting of eight (Exp 3), 10 (Exp 4–8), or 12 (Exp 1, 2) cuttings.

Time course of expression of hormone-related genes in the stem base analyzed by a targeted microarray

To characterize the hormonal response of the two species to cutting excision, we first monitored the transcription of auxin-, JA-, and SL-related genes, as well as genes putatively controlling the interaction of these hormones, in the stem base of the cuttings using a phytohormone-targeted microarray. The intensive rooting of both species observed after 16 to 18 days (Figure 2) reflected a temporal progression of AR formation similar to that of histologically characterized P. hybrida Mitchell, which also showed intensive rooting after 16 days under the same conditions (Klopotek et al., 2010). Thus, a similar timing of the rooting phases in P. axillaris and P. inflata can be expected. According to the phase characterization of P. hybrida Mitchell (Klopotek et al., 2010), the transcriptome of the stem bases of P. axillaris and P. inflata was analyzed during the early induction phase (0.5 and 2 hpe), the central root induction phase (24 hpe), and the expected start of root initiation (72 hpe), and was compared with the respective transcriptome at 0 hpe, the time of excision. The lists, numbers, and ratios of up- and downregulated genes per species and time point for different gene categories and families are compiled in Supplementary Table S3.

The absolute and relative numbers of up- and downregulated genes of different hormonal categories in the stem base of the two species are illustrated in Figures 3, 4. In both species, genes of the JA and hormonal interaction categories showed upregulation as early as 0.5 hpe and reached numbers at 2 hpe that were at or near the maximum level reached over the analyzed period. This was followed by a slight decline in the number of upregulated genes until 24 hpe, before the absolute and relative numbers increased again until 72 hpe (Figures 3A, B). P. inflata reached higher numbers and ratios of upregulated JA-related genes than P. axillaris at 0.5, 24, and 72 hpe. Only a few genes of the JA- and interaction category were downregulated. SL-related genes showed almost exclusive downregulation in both species. This applied to genes of the carotenoid cleavage dioxygenase (CCD) family and the carotenoid isomerase gene D27 (Supplementary Table S3), which control SL biosynthesis (Al-Babili and Bouwmeester, 2015). The highest numbers of DEGs were found in the auxin category. In both species, the numbers of up- and downregulated auxin-related genes increased over time, reaching the highest values at 72 hpe. At 24 and 72 hpe, more auxin-related genes were upregulated in P. inflata than in P. axillaris. At 0.5 and 24 hpe, more auxin-related genes were downregulated in P. axillaris than in P. inflata, whereas the opposite pattern was found at 2 and 72 hpe. Considering the overall dynamics of gene transcription of the four hormonal categories, genes of the JA and interaction categories appeared as early responders, showing approximately the maximum number of upregulated genes already before the most comprehensive response of auxin-related genes was recorded. Petunia inflata displayed more frequent upregulation of JA- and auxin-related genes than P. axillaris.

Figure 3

Figure 3. Number and ratio of up- and downregulated genes (DEGs) of different hormonal categories (A, B) and of JA-related genes (C–F) in the stem base of P. axillaris and P. inflata in response to cutting excision over time. Microarray data. Genes that showed a significantly different expression at the specified time point compared to the initial expression at 0 hours post excision (hpe) were counted. Criteria: t-test (p < 0.05) and log2 FC > 2 or < −2. Sample size (n) = 3, each sample consisting of material from 10 or 16 cuttings for 24 and 72 hpe or 0, 0.5, and 2 hpe, respectively.

Figure 4

Figure 4. Number and ratio of up- and downregulated auxin-related genes (DEGs) of different categories (A-H) in the stem base of P. axillaris and P. inflata in response to cutting excision over time. Microarray data. Genes that showed a significantly different expression at the specified time point compared to the initial expression at 0 hours post excision (hpe) were counted. Criteria: t-test (p < 0.05) and log2 FC > 2 or < −2. Sample size (n) = 3, each sample consisting of material from 10 or 16 cuttings for 24 and 72 hpe or 0, 0.5, and 2 hpe, respectively.

Among the genes putatively controlling JA biosynthesis, genes of the lipoxygenase (LOX), the allene oxide synthase (AOS), the allene oxide cyclase (AOC), and the 12-oxophytodienoic acid (OPDA) reductase (OPR) families were upregulated in both species at 0.5 and 2 hpe, and in P. inflata, also at the later time points (Figures 3C, D). In P. inflata, five and two LOX genes were upregulated at 0.5 and 2 hpe, respectively. By contrast, P. axillaris showed only one LOX gene upregulated at 0.5 hpe, but exhibited an exclusive downregulation of four LOX genes, one AOS gene, and one OPR gene at 24 and 72 hpe. At 2 hpe, both species showed upregulation of all AOC genes, two in P. axillaris and one in P. inflata. In P. axillaris, two genes that constituted 50% of the analyzed JA-amino acid synthetase (JAR) family and control the conversion of JA into the biologically active conjugate jasmonoyl-isoleucine (JA-Ile), were upregulated at 2 hpe, whereas in P. inflata, only one JAR gene showed such a response (Figures 3E, F). Corresponding to the higher number of upregulated LOX and AOS genes in P. inflata, this species also showed a higher number of upregulated jasmonate zim domain (JAZ) genes at all time points (Figures 3E, F). JAZ proteins function as repressors of JA signaling; however, their genes also belong to the early JA-responsive genes (Wasternack and Hause, 2013). One gene of the basic helix–loop–helix transcription factor myelocytomatosis 2 (MYC2), which acts as a positive regulator of the JA signaling and also belongs to the early JA-responsive genes, was upregulated at 0.5 hpe in each species, before two novel interactor of JAZ (NINJA) genes were upregulated at 24 hpe only in P. axillaris.

Most genes that putatively control auxin biosynthesis were downregulated in both species after excision of the cuttings (Figure 4A). This applies to one anthranilate synthase (ASA) gene downregulated in P. inflata at 72 hpe, two tryptophan amino transferase (TAR) genes downregulated in P. inflata at 2, 24, and 72 hpe, and up to four and five YUCCA genes, downregulated in P. axillaris and P. inflata, respectively (Figures 4A, 5A). The only exceptions from this response were the upregulation of one ASA gene in P. axillaris at 0.5 hpe and the upregulation of one TAR and one YUCCA gene (FMO-GC-OX-like 7) in P. inflata at 2 and 72 hpe, respectively (Figures 4A, 5A). However, while auxin biosynthesis was mainly downregulated at the transcriptome level, excision of cuttings stimulated a fast and strong upregulation of ILR/ILL genes that encode IAA-amino acid amidohydrolases, in both species (Figure 4A). This applied to one ILR1-like2 and one ILR1-like6 gene in both species, and additionally to one ILR1-like4 gene in P. axillaris (Figure 5B).

Figure 5

Figure 5. Heat maps showing the differential expression of genes of the YUCCA family (A), the ILL-and ILR-like family (B), and the PIN and PIN-like family (C, D) in the stem base (A–C) and in different cutting sections (D) of P. axillaris and P. inflata in response to cutting excision over time (A–C) or at 24 hours post excision (hpe) (D). Microarray data. Crosses in the boxes indicate a significantly different expression at the specified time point compared to the initial expression at 0 hpe. Criteria: t-test (p < 0.05) and log2 FC > 2 or < −2. Sample size (n) = 3, each sample consisting of material from 10 or 16 cuttings for 24 and 72 hpe or 0, 0.5, and 2 hpe, respectively. UL, upper leaves; BL, basal leaves; SB, stem base, 0.5 cm in length; US, upper stem; SA, shoot apex.

Excision of the cuttings modified the transcription of genes that control auxin transport in the stem base (Figures 4C, D, 5C). Upregulation of PIN and PIN-like genes started at 24 hpe and involved higher gene numbers in P. inflata than in P. axillaris. The number of upregulated PIN and PIN-like genes in P. inflata even strongly increased between 24 and 72 hpe, whereas in P. axillaris, only one gene was upregulated at both 24 and 72 hpe. The upregulation involved PIN5 and PIN6 in both species, while five PIN-like genes were additionally upregulated in P. inflata (Figure 5C). In both species, upregulation of PIN6 started at 24 hpe before PIN5 was upregulated at the start of root initiation (72 hpe). PIN11 was downregulated from 24 hpe onward in P. axillaris and from 2 hpe onward in P. inflata. Auxin-resistant (AUX)/like AUX (LAX) genes, which encode proteins controlling auxin influx, were exclusively downregulated in both species from 24 hpe onward (Figures 4C, D). At this time, more AUX/LAX genes were downregulated in P. axillaris than in P. inflata. Genes encoding the B family of membrane-bound ATP-binding cassette (ABC) transporters (ABCB) were preferentially upregulated in P. axillaris starting at 24 hpe, while P. inflata showed exclusive downregulation at 24 and 72 hpe. At 0.5 hpe, both species showed downregulation of one gene of the PINOID/WAG1/WAG2 family, which may function in intracellular trafficking of PIN proteins.

During the induction phase (0.5 until 24 hpe), expression of Aux/IAA genes that encode auxin repressor proteins was exclusively enhanced in both species (Figures 4E, F). At 2 and 24 hpe, the number and ratio of upregulated Aux/IAA genes were higher in P. inflata than in P. axillaris. At the start of root initiation (72 hpe), in addition to two upregulated Aux/IAA genes, two and three other Aux/IAA genes were downregulated in P. axillaris and P. inflata, respectively (Figures 4E, 6A). Specific ARF genes were upregulated in both species after excision of cuttings, while in P. inflata, this gene family responded earlier and at 2 and 72 hpe involved a higher number and ratio of genes than in P. axillaris (Figures 4E, F). Downregulation of other ARFs was observed at 2, 24, and 72 hpe in P. axillaris but was restricted to 2 hpe in P. inflata (Figures 4E, F). One and two genes that showed homology to ARF11 were upregulated from 2 hpe onward in P. axillaris and from 0.5 hpe onward in P. inflata, respectively, whereas one homologue of ARF9 was downregulated in each species (Figure 6B).

Figure 6

Figure 6. Heat maps showing the differential expression of genes of the Aux/IAA family (A), the ARF family (B), and the LBD family (C) in the stem base of P. axillaris and P. inflata in response to cutting excision over time. Microarray data. Crosses in the boxes indicate a significantly different expression at the specified time point compared to the initial expression at 0 hours post excision (hpe). Criteria: t-test (p < 0.05) and log2 FC > 2 or < −2. Sample size (n) = 3, each sample consisting of material from 10 or 16 cuttings for 24 and 72 hpe or 0, 0.5, and 2 hpe, respectively.

While upregulation of two WUSCHEL-related homeobox (WOX) genes was found only in P. axillaris, both species revealed a comprehensive upregulation of genes of the lateral organ boundaries domain (LBD) family. The number of upregulated genes increased over time between 0.5 and 72 hpe and was higher in P. inflata than in P. axillaris (Figures 4E, 6C). In parallel to the upregulation, fewer LBD genes were downregulated. In P. inflata, the number of downregulated genes increased between 0.5 and 72 hpe, while in P. axillaris, the downregulation was restricted to 24 hpe. In both species, genes showing high homology to LBD41 were upregulated at each of the four time points, whereas genes showing homology to LBD12 were upregulated exclusively at early root initiation (72 hpe). Genes of the Gretchen Hagen 3 (GH3) family that may control conjugation of acidic hormones, particularly of IAA to amino acids, were preferentially upregulated in both species over the period from 0.5 to 72 hpe. At 2 and 24 hpe, the number and ratio of upregulated GH3 genes were higher in P. inflata than in P. axillaris (Figures 4G, H). A large number and ratio of genes of the small auxin up RNA (SAUR) family were differentially expressed in the stem base after excision of the cuttings (Figures 4G, H). At 2 and 24 hpe, P. axillaris showed more downregulated SAUR genes than P. inflata. Genes of the GAI, RGA, and SCR-like (GRAS) family that may control auxin-induced cell reprogramming and differentiation at different levels were exclusively upregulated at 0.5 hpe, then reaching a higher gene number and ratio for P. axillaris (Figures 4G, H). At the following time points, both up- and downregulation were observed. At central root induction (24 hpe) and early root initiation (72 hpe), P. inflata showed a higher number and ratio of upregulated GRAS genes than P. axillaris. Two Cyclin genes that are thought to control cell division were upregulated at the time of root initiation (72 hpe) only in P. axillaris, whereas downregulation of other genes of this gene family was observed in both species between 2 and 72 hpe.

Genes of the ERF family accounted for the largest proportion of regulated genes in the hormonal interaction category (Figure 3A). As illustrated in Figure 7A, 18 and 17 ERF genes were upregulated in the stem base of P. axillaris and P. inflata already at 0.5 hpe. Among these, genes with homology to ERF17 were exclusively upregulated at this time point and reached higher magnitudes of upregulation in P. inflata than in P. axillaris (Figure 7A). During the following time points, upregulation of ERF genes became less pronounced. Among the upregulated genes, homologs of ERF114 and ERF113 that in Arabidopsis respond to JA and have already been related to etiolation-induced adventitious rooting (Lakehal et al., 2020) showed upregulation between 2 and 72 hpe in P. axillaris and P. inflata, respectively.

Figure 7

Figure 7. Heat maps showing the differential expression of genes of the ERF family in the stem base over time (A) and in different cutting sections at 24 hours post excision (hpe) (B) of P. axillaris and P. inflata. Microarray data. Crosses in the boxes indicate a significantly different expression at the specified time point compared to the initial expression at 0 hpe. Criteria: t-test (p < 0.05) and log2 FC > 2 or < −2. Sample size (n) = 3, each sample consisting of material from 10 or 16 cuttings for 24 and 72 hpe or 0, 0.5, and 2 hpe, respectively. UL, upper leaves; BL, basal leaves; SB, stem base, 0.5 cm in length; US, upper stem; SA, shoot apex.

Systemic effects of cutting excision on the expression of hormone-related genes as analyzed by a targeted microarray

At 24 hpe, the time point of central induction, expression of genes was also analyzed in upper cutting parts and related to the initial status at 0 hpe, when cuttings were excised (Supplementary Table S4, Figure 8). To illustrate the magnitude of the systemic effect in relation to the local effect, the data of the stem base are also included in Figure 8. The response of JA-, auxin-, and hormonal interaction-related genes to the excision of cuttings reached the shoot apex as the uppermost cutting part (Figures 8A, B). In the upper stem, the number of upregulated JA-related genes was even as high as in the stem base, with seven versus five upregulated genes for P. inflata compared to P. axillaris. A substantial upregulation of JA-related genes also occurred in upper and lower leaves, with three to four upregulated genes in each species. Almost no downregulation of JA-related genes was observed in the upper cutting parts. As was the case for the stem base, also in upper cutting sections, many DEGs were related to the auxin category (Figure 8A). Numbers and ratios of both up- and downregulated auxin-related DEGs were mostly higher in P. inflata than in P. axillaris (Figures 8A, B). In the latter, the pattern of upregulated genes revealed a strong decline from the stem base to a similarly low level for the upper stem, the upper and lower leaves, and the shoot apex. In P. inflata, a similar apical-directed decline of upregulated auxin-related genes was found, but in leaves, more than twice as many auxin-related genes were upregulated compared to P. axillaris. Similar to the upregulated genes, an apical-directed decline was also found for the downregulated auxin-related genes, which, however, was less strong. Regulation of SL-related genes was restricted to the stem base. The expression pattern of genes of the category hormonal interaction revealed only a weak gradient along the shoot axis, so that up to eight and 10 genes were upregulated in the upper cutting parts of P. axillaris and P. inflata, respectively. Almost no downregulation was observed for this category in the upper cutting parts.

Figure 8

Figure 8. Number and ratio of up- and downregulated genes of different hormonal categories (A-F) in the stem base (SB), the upper stem (US), the two basal leaves (BL), the upper leaves (UL), and the shoot apex (SA) of P. axillaris and P. inflata at 24 hours post excision (hpe). Microarray data. Genes that showed a significantly different expression at the specified time point compared to the initial expression at 0 hpe were counted. Criteria: t-test (p < 0.05) and log2 FC > 2 or < −2. Sample size (n) = 3, each sample consisting of material from 10 or 16 cuttings for 24 hpe or 0 hpe, respectively.

Expression of genes controlling JA biosynthesis and signaling in the upper cutting parts responded to the excision of cuttings, but at different levels of the pathways for the two species (Figures 8C, D). In P. inflata, genes of the AOS family were mostly upregulated and also downregulated in all upper cutting sections except the shoot apex at frequencies similar to the stem base. P. axillaris showed no transcriptional response of AOS genes in the upper cutting parts to the excision, but upregulation and less downregulation of OPR genes in all upper cutting parts. Upregulation of JAZ was restricted to the upper stem in P. inflata and to the upper leaves of P. axillaris. Upregulation of NINJA genes was found in all upper tissues in P. inflata and in the upper stem and both leaf types in P. axillaris. Genes controlling auxin biosynthesis were mostly downregulated in the upper cutting parts in response to the cutting excision, except for one ASA gene that was upregulated in the leaves of P. axillaris (Figures 8E, F). By contrast, expression of auxin transporter genes revealed a species-dependent upregulation in cutting leaves. Five and four PIN/PIN-like genes were exclusively upregulated in basal and upper leaves of P. inflata, respectively, while one other gene of the same category was downregulated in all upper tissues of the same species and in the upper stem and the leaves of P. axillaris. It can be seen from Figure 5D that two genes with homology to PIN-like1 and three other genes with homology to PIN5, PIN-like1, or PIN-like3 were strongly upregulated only in the leaves of P. inflata. In each species, two other genes with homology to PIN11 and to PIN-like3 were downregulated in the upper stem and the leaves, respectively. ERF genes responded in the upper cutting parts to the excision of cuttings. Seven ERF genes in P. axillaris and three in P. inflata were upregulated in the upper cutting parts (Figure 7B). These included ERF114 in P. axillaris and ERF113 in P. inflata (Figure 7B). In each species, two other ERF genes showed downregulation in the upper cutting parts.

Validation of the targeted microarray by RT-qPCR

Considering our focus on auxin, for validation of the microarray, we selected seventeen genes that control auxin biosynthesis, transport, signaling, and downstream response, analyzed the transcript levels in selected samples from P. axillaris or P. inflata by RT-qPCR, and compared the results with microarray data. High correspondence and significant correlation between both methods revealed the high quality of the microarray analysis (Supplementary Figure S1). Lower fold changes for ASA2, IAA20-like, and SAUR68-like determined by RT-qPCR, when compared to the microarray, even indicate that the microarray was more sensitive than RT-qPCR. This conclusion is supported by the expression levels determined by both methods (Supplementary Figure S2). These three genes, particularly SAUR68-like, revealed relatively low signal levels when analyzed by RT-qPCR.

Plant hormone levels as affected by time after excision and cutting section

Corresponding to the analysis of transcription of genes putatively controlling plant homeostasis, signaling, and response, phytohormone concentrations were analyzed in the stem base of both species over time after excision and also in the upper cutting sections at 0 and 24 hpe, the time point of central root induction. In addition to the most important physiologically active auxin, IAA, JA, as well as its precursor OPDA, its physiological conjugate JA-Ile, and diverse cytokinins, which have important antagonistic functions against auxin, were analyzed. Furthermore, salicylic acid (SAL) was included because SAL has recently been found as a new hormonal player affecting AR formation in explants from Arabidopsis and cucumber while interacting with auxin (Dong et al., 2020; Tran et al., 2023). Strigolactones were not analyzed because SLs are extremely difficult to quantify in shoot tissues.

Figure 9 shows hormone dynamics and IAA/cytokinin ratios in the stem base of P. axillaris and P. inflata after excision. In both species, JA peaked sharply at 0.5 hpe before dropping to baseline by 24 hpe, with P. axillaris showing nearly double the peak level of P. inflata (Figure 9A). OPDA followed a similar pattern, but P. axillaris reached over triple the peak level of P. inflata and maintained higher levels throughout (Figure 9B). JA-Ile, generally lower than JA, mirrored the pattern of JA but reached equally high peak levels in both species at 0.5 hpe (Figure 9C). P. axillaris showed slightly, but significantly, higher JA-Ile levels at 0, 2, and 24 hpe. IAA levels in the stem base were about three times higher in P. inflata than in P. axillaris at the time of cutting excision and remained elevated until root initiation at 72 hpe (Figure 9D). IAA rose until 2 hpe and then declined below initial levels. Trans zeatin (tZ), cis zeatin (cZ), isopentenyladenosine (IPR), and trans zeatin riboside (tZR) increased strongly in P. inflata after 24 hpe, reaching higher levels than P. axillaris (Figures 9E–I), while isopentenyladenine (IP) showed little variation (Figure 9G). IAA/cytokinin ratios mirrored IAA trends, peaking at 2 hpe and thereafter decreasing to a minimum at 72 hpe, then revealing only small interspecific differences (Figures 9J–L). IAA correlated strongly (B > 0.83) with these ratios (Figure 10A).

Figure 9

Figure 9. Concentrations of phytohormones in cutting parts of P. axillaris and P. inflata. Temporal course of JA (A), OPDA (B), JA-Ile (C), IAA (D), tZ (E), cZ (F), IP (G), tZR (H), IPR (I), the ratio of IAA/tZ (J), the ratio of IAA/(tZ + IP) (K), the ratio of IAA/(tZ + cZ + IP + tZR + IPR) (L), and the level of SAL (M) in the stem base. Asterisks indicate significant differences between the two species at the specified time points (t-test, p < 0.05). t and T indicate values that are significantly different from the values at 0 hours post excision (hpe) for P. axillaris and P. inflata, respectively (ANOVA, Tukey test, p < 0.05). Sample size (n) = 3, each sample consisting of material from 10 or 16 cuttings for 24 and 72 hpe or 0, 0.5, and 2 hpe, respectively. For abbreviations, see the text.

Figure 10

Figure 10. Linear regressions calculated between IAA in the stem base as independent variable and different IAA/cytokinin ratios as dependent variable, using combined data of P. axillaris and P. inflata from all time points (n = 30) (A). Distribution of IAA (B), IP (C), SAL (D), and JA-Ile (E) between the different cutting parts at 0 h and 24 hours post excision (hpe). Asterisks indicate significant differences between the two species at 0 hpe. Crosses indicate significant differences between the two species at 24 hpe. Each sample consisted of material from 10 or 16 cuttings for 24 or 0 hpe, respectively.

The analysis of the phytohormone-related transcriptome already indicated that excision of cuttings modified plant hormone metabolism at the whole-cutting level. Therefore, we also analyzed plant hormone concentrations at 0 and 24 hpe in the upper cutting parts in addition to the stem base. The results highlight a species-dependent distribution of certain plant hormones along the shoot axis that was additionally affected by the time after excision. In both species, the IAA levels at 0 hpe increased in basipetal direction along the shoot axis, showing the lowest levels in the leaves and an increase from the shoot apex toward the stem base (Figure 10B). However, this gradient was much stronger in P. inflata than in P. axillaris. Furthermore, the basipetal increase of IAA along the shoot axis in P. inflata remained at a high level until central root induction (24 hpe), whereas in P. axillaris, the gradient almost completely disappeared by 24 hpe (Figure 10B). At both time points, the gradients of IAA concentration between basal leaves or the upper leaves and the stem base were significantly higher in P. inflata compared to P. axillaris (Supplementary Table S5).

Concerning the other hormones, the most temporally stable difference between the two species in the upper cutting parts was a significantly lower level of IP in leaves for P. inflata compared to P. axillaris (Figure 10C). Trans-zeatin increased in the upper cutting sections between 0 and 24 hpe, and at 24 hpe, it only differed between the species in the upper stem, with lower levels in P. inflata (Supplementary Table S6). The concentrations of tZR were highest in the shoot apex and the upper stem, while P. inflata revealed significantly higher levels than P. axillaris in all stem sections at 0 hpe and in all cutting sections at 24 hpe (Supplementary Table S6). At 0 hpe, SAL concentrations in P. inflata showed the highest levels in the leaves and were higher than in P. axillaris in all cutting sections (Figure 10D). The SAL gradient between leaves and the stem base of P. inflata became inverted between 0 and 24 hpe. Nevertheless, P. inflata maintained higher SAL levels in the leaves than P. axillaris. JA-Ile was similarly low in all tissues of both species at 0 hpe but showed an apical increase along the shoot axis only in P. axillaris at 24 hpe (Figure 10E). Furthermore, P. inflata revealed lower JA levels in most tissues at 24 hpe than P. axillaris (Supplementary Table S6).

Correlations between leaf hormone levels and the IAA gradient to the stem base were analyzed (Supplementary Table S7). Stem-base IAA was unrelated to leaf IAA levels but strongly correlated with the leaf–stem gradient (r = 0.997), indicating that transport intensity rather than leaf auxin levels determined stem-base IAA. The IAA gradient was negatively correlated with leaf IP, JA, OPDA, and JA-Ile (Figures 11A, B, Supplementary Table S7) but positively correlated with leaf SAL (Figures 11C, D). However, for JA, OPDA, and JA-Ile, data distributions were uneven (Supplementary Figure S3).

Figure 11

Figure 11. Linear regressions calculated between IP in the basal leaf (A), IP in the upper leaf (B), SAL in the basal leaf (C), and SAL in the upper leaf (D) as the independent variables and the IAA concentration gradient between the respective leaf and the stem base as the dependent variable. Combined data of P. axillaris and P. inflata from 0 and 24 hours post excision (hpe) (n = 12). Each sample consisted of material from 10 or 16 cuttings for 24 or 0 hpe, respectively.

Response of adventitious rooting of P. axillaris and P. inflata to stem base-targeted IAA application and to blocking of PAT from the upper shoot

The transcriptome data, as well as the phytohormone levels in the stem base, indicated that P. inflata, which showed a higher rooting potential when compared with P. axillaris, benefits from the locally higher IAA levels that could evoke enhanced auxin signaling at the levels of Aux/IAA, ARF, LBD, and PIN gene transcripts during the induction phase, thereby promoting AR formation. If this were the case, AR formation in P. axillaris should benefit more from external IAA supply to the stem base than that in P. inflata. To test this hypothesis, in three replicative experiments, cuttings of both species were rooted under standard climate chamber conditions while being exposed to increasing concentrations of IAA for the first 72 h after excision. With the start of root initiation at 72 hpe, cuttings were replanted in perlite until rooting was assessed on day 16 after excision. In all experiments, cuttings of P. inflata produced fewer and shorter ARs compared with P. axillaris when no IAA was supplied (Figures 12A–F). However, root number and length of P. axillaris were significantly enhanced by the application of 5 mg L⁻¹ IAA, whereas these parameters were not significantly affected by the same dosage in P. inflata (experiments 1 and 2, Figures 12A–D). Additionally, the application of 10 mg L⁻¹ IAA resulted in a strong increase in root numbers and length in P. axillaris in all three experiments compared to the controls, whereas such an increase was nonsignificant (Exp 1, Figures 12A, B) or much smaller (Figures 12C–F) for P. inflata. By contrast, AR formation of P. inflata apparently benefited more from higher IAA dosages than P. axillaris. Thus, in experiments 2 and 3, the root number of P. inflata increased or remained at a similar level when IAA supply was increased from 10 to 50 mg L⁻¹, respectively, whereas the root number of P. axillaris was maintained or even slightly decreased (Figures 12C, E). Further increase of IAA from 50 to 150 mg L⁻¹ decreased the root number of P. axillaris but had no negative effect on P. inflata (Figure 12E). Increasing IAA supply above 10 mg L⁻¹ reduced root length of P. axillaris in all three experiments, although not significant at the individual experimental level, while root length of P. inflata remained at the 10 mg L⁻¹ level or even slightly increased with the 50 mg L⁻¹ dosage in experiment 2 (Figures 12B, D, F).

Figure 12

Figure 12. Rooting response of P. axillaris and P. inflata to applications of different dosages of IAA applied between 0 and 72 hours post excision (A–H) and to continuous lanolin-mediated applications of naphtylphatalamic acid (NPA) (I). Number (A, C, E) and length (B, D, F) of formed adventitious roots per planted cutting in three independent experiments. Mean auxin response of root number (G) and root length (H) in terms of difference to the IAA-free control (0 mg L⁻¹ IAA), calculated over the three experiments. Root number as affected by different dosages of NPA (I). Columns and whiskers show the mean values ± SE. Numbers above the columns in (I) additionally show the mean values. Different upper- or lowercase letters indicate significant differences between the IAA and NPA dosages for P. axillaris or P. inflata, respectively (ANOVA, Tukey test, p < 0.05, n = 4 [Exp 1–3], n = 3 [Exp. 4, 5], each n consisting of 10 cuttings).

Figures 12G, H show that the interspecific difference in auxin response depended on auxin concentration, depicting the mean absolute differences in root number and length at various IAA levels relative to the IAA-free control across three experiments. P. axillaris showed a higher increase in root number and length than P. inflata up to a dosage of 10 mg L⁻¹ IAA. With 50 mg L⁻¹ IAA, the increase in root number was only slightly higher in P. axillaris than in P. inflata, whereas the increase in root length was already lower in P. axillaris. With 150 mg L⁻¹ IAA, P. axillaris showed a smaller increase in root number and length than P. inflata.

The exclusive upregulation of auxin transporter genes in P. inflata, together with a stronger IAA gradient between the leaves and the stem base compared to P. axillaris, supports the conclusion that the cutting leaves in Petunia are important auxin source organs, from which IAA is transported to the stem base, where it can induce the formation of ARs. To analyze the contribution of PAT-controlled auxin influx from cutting parts above the basal stem, in particular the leaves, into the stem base, two experiments were conducted with P. inflata and P. axillaris, respectively, using the PAT blocker NPA. Lanolin rings containing NPA at two concentrations or pure lanolin were placed around the bases of the two basal leaves and the stem immediately above the two basal leaves to interrupt, or not interrupt, the PAT-derived auxin flow from the upper shoot, including all leaves, to the stem base (Figure 1B). In both species, NPA application at 10 mg L⁻¹ lanolin and 30 mg L⁻¹ lanolin almost completely and completely inhibited AR formation, respectively (Figure 12I). The results document that the PAT-dependent basipetal transport of IAA from the upper shoot, including the leaves, to the stem base is a bottleneck of adventitious rooting in both species.

Discussion

P. inflata has a higher rooting capacity than P. axillaris, but the realization of rooting also depends on donor plant factors

Both species rooted efficiently and formed adventitious roots rapidly without exogenous auxin, but P. inflata generally showed higher rooting capacity (Figure 2). In some experiments, however, P. axillaris exhibited stronger rooting (Figures 2, 12). As donor plants were cultivated under variable greenhouse conditions, while rooting always occurred under identical climate chamber conditions, these findings suggest that rooting is genetically controlled but also influenced by the physiological state of the donor plants. Rooting of P. hybrida Mitchell cuttings, for instance, depends on nitrogen supply to donor plants without affecting auxin homeostasis (Zerche et al., 2016; Yang et al., 2019). Likewise, photosynthesis of cuttings, influencing carbohydrate supply for root growth (Rapaka et al., 2005), is modified by donor plant light conditions (Klopotek et al., 2012). Since nitrogen nutrition was optimized based on repeated substrate analysis in this study, variations in light, temperature, and plant age likely affected rooting, with P. inflata apparently less resilient than P. axillaris. Nevertheless, the data as a whole indicate a higher rooting capacity of P. inflata, since the minimum level of rooting that could be reached across all experiments was higher in this species (Figures 2, 12).

Both species reveal an early activation of the jasmonic acid pathway in the stem base during the root induction phase, which is further linked to the ERF family

The phytohormone-targeted microarray proved to be a reliable and valuable tool for monitoring hormone-related transcriptomes in both Petunia species (Supplementary Figures S1, S2). One of the earliest responses in the stem base after cutting excision was the upregulation of genes controlling JA biosynthesis and signaling at 0.5 hpe, followed by genes for JA-Ile biosynthesis at 2 hpe (Figures 3C–F). This was accompanied by a marked increase in JA and JA-Ile levels in the same tissue, peaking at 0.5 hpe (Figures 9A, C). While JA levels peaked higher in P. axillaris, JA-Ile reached similar maxima in both species. Considering that JAZ genes are among the earliest JA-induced genes (Wasternack and Song, 2017), the stronger and more persistent JAZ activation in the stem base (Figure 3E) and upper stem (Figure 8C) of P. inflata indicates enhanced JA signaling compared with P. axillaris. Upregulation of JA biosynthesis and signaling is a typical response to wounding (Wasternack and Song, 2017). A similarly early, transient JA accumulation in the stem base was reported for P. hybrida and pea cuttings (Ahkami et al., 2009; Rasmussen et al., 2015). Although JA can inhibit etiolation-induced AR formation in Arabidopsis hypocotyls, involving changes in auxin and cytokinin homeostasis and signaling (Gutierrez et al., 2012; Lakehal et al., 2019; Dob et al., 2021), accumulating evidence supports its positive regulatory role in cuttings. Suppression of wound-induced JA synthesis by AOC silencing reduced AR formation in P. hybrida cuttings (Lischweski et al., 2015), whereas short-term JA application to pea cuttings promoted rooting (Rasmussen et al., 2015). These findings support the hypothesis that early activation of the JA pathway contributes to AR formation in P. axillaris and P. inflata and may also partially underlie the higher rooting capacity of P. inflata. The stimulation of the JA pathway extended beyond the stem base, reaching upper leaves and the shoot apex at 24 hpe (Figures 8A, B, 10E, Supplementary Table S6), suggesting JA involvement in systemic processes supporting AR formation.

ERF genes showed frequent and strong upregulation, starting in the stem base at 0.5 hpe (Figure 7A) and, in specific cases, also in upper cutting sections at 24 hpe (Figure 7B). In Arabidopsis, depending on the specific ERF gene, expression can be regulated by ethylene as well as by other plant hormones such as JA, ABA, and SAL (Heyman et al., 2018). ERF genes have various functions, including wound response, tissue repair, stress tolerance, pathogen resistance, and plant development (Heyman et al., 2018; Wu et al., 2022). In this study, both species showed local upregulation in the stem base of ERF1, ERF2, ERF4, ERF5, ERF13, ERF17, and ERF25. In Arabidopsis, ERF1 integrates ethylene and JA signals (Lorenzo et al., 2003) and regulates primary and lateral root development involving upregulation of auxin transporters (Mao et al., 2016; Zhao et al., 2023). ERF2 controls root growth in rice and affects responses to ethylene, ABA, auxin, and cytokinins (Xiao et al., 2016). Genetically engineered upregulation of auxin biosynthesis in apple rootstocks enhanced ERF5 expression in both rootstocks and scions (Zhai et al., 2021), and overexpression of ERF5 in Dendrobium orchid promoted regeneration of protocorm-like bodies (Zeng et al., 2023). In this study, P. axillaris showed strong upregulation of ERF114 in the stem base from 24 hpe onward (Figure 7A) and in the upper stem at 24 hpe (Figure 7B). P. inflata exhibited local upregulation of ERF113, a close homolog of ERF114, between 2 and 72 hpe (Figure 7A) and in all upper cutting parts except the shoot apex at 24 hpe (Figure 7B). In Arabidopsis, ERF113 is responsive to JA, SAL, ABA, and ethylene (Heyman et al., 2018). Both ERF113 and ERF114 are rapidly induced by wounding (Ikeuchi et al., 2017) and play roles in etiolation-induced AR formation in hypocotyls of Arabidopsis seedlings, where JA acts as a negative regulator (Lakehal et al., 2020). These results indicate that early upregulation of distinct ERF genes, such as ERF1, ERF2, ERF5, and particularly ERF113/ERF114, contributes to AR induction in P. axillaris and P. inflata, possibly acting downstream of JA and ethylene and upstream of auxin.

Early local upregulation of ILR/ILL genes and PINOID-mediated rearrangement of intracellular auxin distribution seem to contribute to the auxin homeostasis in the stem base of P. inflata and P. axillaris

The highest number of DEGs occurred in the auxin category (Figures 3A, B). Expression data from the stem base showed predominant downregulation of genes controlling auxin biosynthesis, particularly YUCCA genes (Figures 4A, B), contrasting sharply with the upregulation of several YUCCA genes in Arabidopsis leaf explants during AR formation (Chen et al., 2016). Both species, however, showed rapid and strong upregulation of ILR1-like2 and ILR1-like6, and P. axillaris additionally upregulated ILR1-like4 (Figures 4A, B, 5B). ILR/ILL genes encode auxin-conjugate hydrolases that have been extensively characterized in Arabidopsis. In this plant, specific genes exhibit distinct but overlapping substrate specificities for conjugates between IAA or IBA and individual amino acids (Smolko et al., 2018), and hydrolases of the ILR1-like family identified regulate auxin homeostasis in the endoplasmic reticulum (ER) (Carranza et al., 2016). ILR genes can be induced by wounding and JA. In this context, ILR1, ILL6, and IAR3, members of the ILR1-like family (Campanella et al., 2003), were upregulated in Arabidopsis by wounding and JA and not only controlled auxin-conjugate hydrolysis but also reduced JA signaling via hydrolysis of JA-Ile (Zhang et al., 2016b). These findings suggest that early upregulation of ILR1-like2, ILR1-like4, and ILR1-like6 contributes to the increase in IAA at 2 hpe and possibly to the simultaneous decrease in JA-Ile in P. axillaris and P. inflata (Figures 9C, D), thereby influencing AR formation.

PINOID protein serine/threonine kinases and their close homologs WAG1 and WAG2 phosphorylate PIN proteins, directing them to the apical cell side, whereas PP2A phosphatase antagonistically dephosphorylates PINs, targeting them basally (Friml et al., 2004; Michniewicz et al., 2007; Adamowski and Friml, 2015; Armengot et al., 2016). In this study, one gene of the PINOID/WAG1/WAG2 family was downregulated at 0.5 hpe in each species (Figure 4C; Peaxi162Scf00333g00017.1, Peinf101Scf00423g01001.1 in Supplementary Table S3). Both genes share high identity with the PINOID gene A4A49_10797 (protein ID AMO02498.1) of Nicotiana attenuata. Overexpression of a PINOID gene in rice delayed crown root formation, a type of AR formed on intact monocot seedlings (Morita and Kyozuka, 2007). In Arabidopsis, AR formation in the hypocotyl of pre−etiolated flooded seedlings was strongly inhibited in the triple mutant pid14 wag1 wag2, even when external auxin was applied, indicating that proper PIN phosphorylation by these kinases is essential for AR establishment (Da Costa et al., 2020). These findings suggest that early downregulation of the A4A49_10797 homolog in the stem bases of P. axillaris and P. inflata may enhance basipetal IAA transport, directing auxin toward the founder cells initiating AR formation.

The preferential downregulation of CCDs and D27, which control SL biosynthesis, observed in P. axillaris and P. inflata, is consistent with similar findings in P. hybrida Mitchell (Bombarely et al., 2016). These results support the hypothesis that SL downregulation contributes to AR formation in both species, possibly by reducing the inhibitory influence of SL on PAT and AR initiation, as reported in Arabidopsis and pea (Rasmussen et al., 2012; Shinohara et al., 2013; Koltai, 2014).

Rooting of P. inflata benefits from a higher auxin accumulation in the stem base that determines a higher auxin/cytokinin ratio and promotes the auxin signal transduction during AR induction

In both species, IAA increased after cutting excision until 2 hpe and then decreased to below the initial level, with consistently higher concentrations in P. inflata (Figure 9D). Changes in auxin transporter expression in the stem base were mainly detected at 24 hpe, with PIN/PIN-like genes more frequently upregulated in P. inflata (Figures 4C, D, 5C). This suggests that transporter gene regulation in the stem base was a consequence of elevated IAA levels rather than their cause. In hypocotyls of pre−etiolated Arabidopsis, auxin−responsive rearrangement of transporter expression has been shown to drive cell reprogramming and subsequent differentiation during adventitious rooting (Della Rovere et al., 2013). Similarly, auxin transporters displayed auxin−responsive, phase−specific expression profiles in the stem bases of tomato and olive cuttings (Guan et al., 2019; Velada et al., 2020). Both Petunia species showed upregulation of PIN6 at 24 hpe during AR induction and of PIN5 at 72 hpe, corresponding to the expected onset of AR initiation (Figure 5C). In Arabidopsis, PIN5 encodes a functional auxin transporter localized to the ER and is required for auxin−mediated development (Mravec et al., 2009). PIN6 localizes to both the ER and plasma membrane, mediating auxin transport across the plasmalemma and regulating intracellular auxin homeostasis (Cazzonelli et al., 2013; Simon et al., 2016). Overexpression of PIN6 promoted AR formation in intact and de−rooted Arabidopsis seedlings (Cazzonelli et al., 2013; Simon et al., 2016). Only P. inflata exhibited upregulation of PIN-like1 and/or PIN-like3 genes, with one PIN-like3 gene already induced at 24 hpe (Figure 5C). These findings highlight PIN6 as a key auxin transporter for AR induction in Petunia, supported in P. inflata by PIN-like3.

Consistent with the higher maximum IAA level in the stem base at 2 hpe (Figure 9D) and more frequent upregulation of auxin transporters thereafter (Figure 5D), P. inflata also showed stronger upregulation of Aux/IAA and ARF genes controlling auxin signaling (Figures 6A, B). Aux/IAA proteins act as auxin repressors of ARF proteins, which are positive regulators directly controlling transcription of auxin-responsive genes such as LBD or GH3 (Leyser, 2018). Auxin-induced degradation of specific Aux/IAA proteins subsequently enhanced transcription of their own encoding genes (Krogan and Berleth, 2015). Thus, the more frequent upregulation of Aux/IAA genes in P. inflata (Figure 4E) likely reflects stronger auxin perception, consistent with its higher IAA concentration (Figure 9D). In this study, one and two genes homologous to ARF11 were strongly upregulated between 2 and 72 hpe in the stem base of P. axillaris and between 0.5 and 72 hpe in P. inflata, respectively (Figure 6B). In Arabidopsis, mutation of ARF11 reduced lateral root number and length (Yamauchi et al., 2024). One homolog of ARF9 was downregulated between 2 and 72 hpe in P. axillaris and at 2 hpe in P. inflata. In transgenic tomato plants, increasing or decreasing ARF9 transcript levels suppressed or enhanced cell division during early fruit development, respectively, demonstrating an inhibitory function of ARF9 in cell division (de Jong et al., 2015). A similar inhibitory role may apply to ARF9 during early cell division events of AR formation in P. inflata and P. axillaris, suggesting that upregulation of ARF11 combined with downregulation of ARF9 supports AR formation in both species.

Genes of the LBD family, which are regulated by auxin and other phytohormones such as jasmonate and ethylene (Majer and Hochholdinger, 2011; Rong et al., 2024), responded strongly to cutting excision in both Petunia species. More LBD genes were upregulated in P. inflata than in P. axillaris, with differences apparent from 2 hpe onward (Figure 6C). In both species, one homolog of LBD41 was upregulated from 0.5 hpe throughout the sampling period until 72 hpe (Figure 6C). At 24 hpe and 72 hpe, LBD41 upregulation was stronger in P. inflata (M-values 5.27 and 7.03) than in P. axillaris (M-values 3.54 and 5.88) (Supplementary Table S3). Consistent with our findings, LBD41 was also induced in the stem base of de-rooted mung bean seedlings at 6 and 24 hpe, and hydrogen peroxide stimulation of AR formation further enhanced its expression (Li et al., 2017). In both Petunia species, homologs of LBD1 were upregulated from 2 hpe onward (Figure 6C). While the role of LBD1 in adventitious rooting remains unclear, it has been linked to secondary growth and cambium differentiation (Rong et al., 2024). In rice, LBD1–8 acts downstream of auxin signaling and is highly expressed in the cortex and lateral root primordia, with lateral root formation depending on its expression (Yamauchi et al., 2019). In both Petunia species, one homolog of LBD16 was upregulated during the induction phase (24 hpe) and at AR initiation (72 hpe) (Figure 6C). Similarly, LBD16 is induced in Arabidopsis leaf explants during early AR induction, regulating AR organogenesis in an auxin-dependent manner (Liu et al., 2014, 2018). Furthermore, overexpression of an apple homolog of LBD16 in tomato increased the number of ARs (Wang et al., 2023). Overall, the data indicate more frequent and stronger LBD gene upregulation in the stem base of P. inflata compared with P. axillaris, consistent with its higher IAA levels, stronger auxin signaling activation, and greater rooting capacity. In both species, LBD1, LBD16, and LBD41 appear to play major roles in excision-induced AR formation, likely acting downstream of auxin and possibly other phytohormones.

In addition to the early upregulation of homologs of JAR1 (Figures 3E, F), which is GH3.11 in Arabidopsis (Wasternack and Song, 2017), both Petunia species showed preferential upregulation of other GH3 genes in the stem base (Figure 4G). At 2 and 24 hpe, more GH3 genes were upregulated in P. inflata than in P. axillaris. Many GH3 proteins act as IAA−amidosynthetases and/or JA−amidosynthetases (Jez, 2022), so GH3 upregulation may have contributed to the reduction of free IAA and JA toward AR initiation (72 hpe in Figures 9A, D). Considering the inhibitory roles of high JA and IAA levels during AR initiation (Da Costa et al., 2013), GH3 induction may thus facilitate final AR formation in P. axillaris and P. inflata.

Differentially expressed SAUR genes (Figure 4G) are also likely to play crucial roles in AR formation in both species. SAUR proteins are transcriptionally induced by auxin in various plant species and are involved in hormone−mediated development (Ren and Gray, 2015), although auxin−induced SAUR repression has also been observed (Paponov et al., 2009). In shoots, specific SAURs control cell expansion by targeting PP2C.D phosphatases (Spartz et al., 2014; Ren and Gray, 2015; Fendrych et al., 2016; Ren et al., 2018). Recent studies in Arabidopsis, cucumber, and sweet potato have shown that specific SAUR proteins can also respond to jasmonate, can promote lateral root and AR formation by stimulating cell expansion, and, in some cases, act upstream of auxin by increasing auxin levels (Yin et al., 2020; Luan et al., 2023; Zhou et al., 2024).

The higher rooting capacity of P. inflata versus P. axillaris can be explained by the higher IAA levels, the higher IAA/cytokinin ratio, the associated stronger auxin signaling at the levels of Aux/IAA and ARF transcription, and the stronger downstream response, as reflected by the higher PIN/PIN-like, GH3, and LBD expression. The idea that the lower endogenous IAA level is a key factor for the lower rooting capacity of P. axillaris is supported by the finding that rooting of this species responded more positively to low IAA dosages of 5 to 10 mg L⁻¹ IAA during root induction than did P. inflata (Figure 12). However, the finding that rooting of P. inflata was stronger enhanced by high IAA dosages of 50 mg and 150 mg L⁻¹, than that of P. axillaris further reveals a higher maximum auxin response capacity for P. inflata. The increase in various cytokinins in the stem base of P. inflata between 24 and 72 hpe suggests a role in establishing new cell clusters—the earliest signs of root initiation in Petunia (Ahkami et al., 2009). In this context, cytokinins are well-known promoters of cell division within the quiescent center of Arabidopsis roots (Schaller et al., 2014).

Rooting of both species is dependent on leaf-derived, PAT-controlled auxin supply, which determines a leaf-stem base auxin gradient, while P. inflata obviously benefits from higher transcription of PIN and PIN-like transporters in the leaves

NPA inhibits auxin efflux from plant cells, likely by affecting PIN- and ABCB-mediated auxin transport (Teale and Palme, 2018). Application of 30 mg L⁻¹ NPA in lanolin around basal leaf bases and the upper stem of Petunia cuttings (Figure 1) completely prevented rooting in both species (Figure 12I). This demonstrates that auxin influx from the upper cutting parts, including leaves, into the stem base is essential for AR formation of both species. P. inflata showed a steeper IAA gradient between leaves, upper stem, and stem base (Figure 10B, Supplementary Table S5), associated with exclusive upregulation of PIN5 and PIN-like genes in leaves (Figure 5D). Auxin transporters at the ER, such as PIN5 and PIN-like, have been suggested to reduce auxin availability for signaling and export from the cell by sequestration of IAA into the ER (reviewed in Ung, 2024). However, in a recent study by Zheng et al. (2025), virus-induced gene silencing of PIN-like2, which is expressed in the ER, decreased the expression of Aux/IAA and GH3 genes and caused significant reductions in the number and length of formed roots. Further molecular assays in the same study demonstrated that one member of the TGA bZIP transcription factor family, TGA7, directly binds the promoter of PIN-like2 and stimulates its transcription (Zheng et al., 2025). TGA factors are key components in SAL signaling activated by SAL (Seyfferth and Tsuda, 2014).

Leaf SAL and cytokinin levels may control the leaf-derived auxin supply to the stem base of cuttings

Negative correlations were found between leaf IP levels and the leaf–stem base IAA gradient (Figures 11A, B). Cytokinins regulate PIN gene transcription and PIN protein trafficking within cells, enhancing auxin transport in shoots related to shoot branching (Marhavy et al., 2014; Simásková et al., 2015; Waldie and Leyser, 2018). P. inflata cuttings showed higher SAL levels in leaves compared to P. axillaris (Figure 10D), which were positively correlated with the IAA gradient between leaves and stem base (Figures 11C, D). SAL influences auxin transport and adventitious rooting, though its roles are not fully understood. SAL application increased transcripts of four PIN genes in cotton shoots (He et al., 2017) but inhibited rootward [³H-IAA] transport and reduced PIN activity in Arabidopsis roots via hyperphosphorylation (Tan et al., 2020). Continuous SAL supply enhanced AR formation in cucumber hypocotyl cuttings (Dong et al., 2020). However, genetic reduction of SAL biosynthesis in Arabidopsis increased rooting percentage, while SAL treatment reduced it (Tran et al., 2023). Considering that these findings are limited to other plant species, lower IP and higher SAL levels in P. inflata may promote auxin export from leaves by unknown mechanisms, possibly involving activation of PIN-like genes via TGA transcription factors, as previously discussed.

Conclusive model

The data highlight a coordinated activation of the hormonal machinery in the stem base of the two Petunia species during AR induction. This process begins with the activation of JA and ERF transcription factors, followed by auxin, which depends strongly on auxin influx from the upper cutting sections. A conceptual model of these relationships is shown in Figure 13. Wounding triggers early JA accumulation in the stem base, reaching higher levels in P. axillaris and coinciding with enhanced expression of JA biosynthetic genes—OPR and AOC in P. axillaris, and LOX and AOS in P. inflata. Although JAR upregulation is higher in P. axillaris, JA-Ile peaks at similar levels in both species and is perceived via the COI1–JAZ co-receptor complex, reflected by elevated MYC2 and JAZ expression. The stronger JAZ upregulation in P. inflata indicates a more pronounced JA response. Several wound-responsive ERF genes (ERF1, ERF2, ERF5) are induced in both species, while ERF113 and ERF114 are induced in a species-specific manner. The ERF and jasmonate pathways likely interact with the auxin pathway. Jasmonate and ERF activation also occur in the upper cutting parts of P. axillaris, as shown at 24 hpe by transcriptomic and JA-Ile data. During AR induction, PAT directs IAA basipetally from the upper sections to the stem base, with leaves likely serving as auxin sources. Downregulation of CCDs and D27 may promote auxin transport and IAA accumulation by reducing inhibitory SLs. Exclusive upregulation of PIN5 and PIN-like1/3 in P. inflata leaves likely enhances auxin export, resulting in stronger basipetal IAA transport and a steeper IAA gradient, controlled by higher SAL and lower IP levels. Consequently, P. inflata shows increased IAA accumulation and a higher IAA/cytokinin ratio, favoring AR induction. This elevated ratio activates auxin signaling genes regulating IAA perception, signal transduction, and cellular response, leading to feedback changes in IAA distribution. Downregulation of PINOID (A4A49_10797) may favor basal PIN protein accumulation. Stronger induction of Aux/IAA, ARF, GH3, PIN/PIN-like, and LBD genes in P. inflata likely contributes to its higher rooting capacity compared to P. axillaris. Local upregulation of ARF11 and PIN6, downregulation of ARF9, and exclusive induction of PIN-like3 in P. inflata suggest key roles for these genes in AR induction. Upregulation of LBD1, LBD16, and LBD41 in both species supports their function in auxin-dependent AR formation. Specific ILR-like genes may regulate the JA-Ile decrease after 0.5 hpe via deconjugation, facilitating AR initiation.

Figure 13

Figure 13. Conceptual model of hormone action in cuttings of P. axillaris and P. inflata during AR induction (0 until 24 hpe), highlighting important plant hormones, gene families, and genes. In the upper part, the blue- and reddish-shaded halves represent the upper cutting parts of P. axillaris and P. inflata, respectively. There, the increase of JA-Ile between 0 and 24 hpe (applies only to P. axillaris), the difference in IP and SAL levels between the two species, and the regulation of genes in the leaves of the two species are illustrated. The distribution of IAA (mean level calculated between 0 and 24hpe) among the five cutting sections of the two species is indicated by the intensity of green coloring in the separated right and left halves. In the lower part, the dynamics of hormone homeostasis and the regulation of important genes are illustrated for both species. Here, different responses of P. axillaris versus P. inflata or common responses of both species are indicated by blue, red, or black shading and the same coloring of the arrows. Red lines and arrows represent the PAT stream in the cutting. Solid and broken lines and arrows indicate established and putative interrelationships, respectively. Further explanation is given in the legend.

These findings enhance the understanding of hormonal regulation of adventitious rooting and provide a basis for the functional analysis of candidate genes using Agrobacterium-mediated transformation and CRISPR/Cas mutagenesis (Zhang et al., 2016a).

Data availability statement

The original data has now been published in GEO under the accession number: GSE317986.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE317986.

Author contributions

IJ: Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing. SC: Investigation, Methodology, Writing – review & editing. NN: Investigation, Methodology, Writing – review & editing. GUB: Investigation, Writing – review & editing, Writing – original draft. UD: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.

Funding

The author(s) declared financial support was received for this work and/or its publication. This work was supported by funds of the Federal Ministry of Agriculture, Food and Regional Identity (BMLEH) based on a decision of the Parliament of the Federal Republic of Germany via the Federal Office for Agriculture and Food (BLE), grant numbers 2818HSE02 and 2823HSE02.

Acknowledgments

We thank Luca Gewinner and Henrike Thomas for their accurate technical assistance, Florian Peschl and Marina Beuke for their horticultural care of the plants, and Dr. Stefan Ehrentraut and Dr. Samanehsadat Maleki for their help in bioinformatic analysis (all FGK). We also greatly thank Prof. Cris Kuhlemeier, University of Bern, Switzerland, for providing early access to the P. axillaris genome Pax403. The grammar and spelling of the American English text were checked with the help of Perplexity AI, which continuously updates and uses the Sonar family of models built on Llama 3.3 70B (https://www.perplexity.ai/).

Conflict of interest

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.

Generative AI statement

The author(s) declare that Generative AI was used in the creation of this manuscript. The grammar and spelling of the American English text were checked with the help of Perplexity AI, which continuously updates and uses the Sonar family of models built on Llama 3.3 70B (https://www.perplexity.ai/).

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2025.1707238/full#supplementary-material

Glossary

ABCB: B family of membrane-bound ATP-binding cassette (ABC) transporters

AOC: allene oxide cyclase

AOS: allene oxide synthase

AR: adventitious root

ARF: auxin response factor

ASA: anthranilate synthase

AUX: auxin-resistant

Aux/IAA: auxin indole-3-acetic protein

BL: basal leaves

cZ: cis zeatin

dpe: days post excision

ET: ethylene

ERF: ethylene response factor

FMO-GC-OX-like: flavin-monooxygenase glucosinolate-S-oxygenase-like

GH3: Gretchen Hagen 3

GRAS: GRAS, GAI, RGA, and SCR like

hpe: hours post excision

IAA: indole-3-acetic acid

ILL: IAA-leucine resistant-like

ILR: IAA-leucine resistant

IP: isopentenyladenine

IPR: isopentenyladenosine

JA: jasmonic acid

JA-Ile: jasmonoyl-isoleucine

JAR: JA-amino acid synthetase

JAZ: jasmonate ZIM domain

LAX: like AUX

LBD: lateral organ boundaries domain

LOX: lipoxygenase

MYC2: myelocytomatosis 2

NPA: 1-N-naphthylphthalamic acid

NINJA: novel interactor of JAZ

OPR: 12-oxophytodienoic acid reductase

OPDA: 12-oxophytodienoic acid

PAT: polar auxin transport

PIN: PIN-formed

PINOID: protein serine/threonine kinase PINOID

SAL: salicylic acid

SAUR: small auxin up RNA

SA: shoot apex

SB: stem base

SL: strigolactones

TAR: tryptophan amino transferase

tZ: trans zeatin

tZR: trans zeatinriboside

UL: upper leaves

US: upper stem

WOX: WUSCHEL-related homeobox

YUCCA: YUCCA family of flavin monooxygenase

WAG1/WAG2: protein serine/threonine kinases WAG1 and WAG2

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