Autonomous differentiation of transgenic cells requiring no external hormone application: the endogenous gene expression and phytohormone behaviors

The ectopic overexpression of developmental regulator (DR) genes has been reported to improve the transformation in recalcitrant plant species because of the promotion of cellular differentiation during cell culture processes. In other words, the external plant growth regulator (PGR) application during the tissue and cell culture process is still required in cases utilizing DR genes for plant regeneration. Here, the effect of Arabidopsis BABY BOOM (BBM) and WUSCHEL (WUS) on the differentiation of tobacco transgenic cells was examined. We found that the SRDX fusion to WUS, when co-expressed with the BBM-VP16 fusion gene, significantly influenced the induction of autonomous differentiation under PGR-free culture conditions, with similar effects in some other plant species. Furthermore, to understand the endogenous background underlying cell differentiation toward regeneration, phytohormone and RNA-seq analyses were performed using tobacco leaf explants in which transgenic cells were autonomously differentiating. The levels of active auxins, cytokinins, abscisic acid, and inactive gibberellins increased as cell differentiation proceeded toward organogenesis. Gene Ontology terms related to phytohormones and organogenesis were identified as differentially expressed genes, in addition to those related to polysaccharide and nitrate metabolism. The qRT-PCR four selected genes as DEGs supported the RNA-seq data. This differentiation induction system and the reported phytohormone and transcript profiles provide a foundation for the development of PGR-free tissue cultures of various plant species, facilitating future biotechnological breeding.


Introduction
Plants can regenerate a whole new plant from a single cell through dedifferentiation and redifferentiation, which is an ability named totipotency.Artificial control of totipotency in cell and tissue culture has been used for plant conservation, breeding, and scientific research because it accelerates not only plant propagation, but also the production of genetically modified plants.Cell differentiation is controlled by the external application of plant growth regulators (PGRs), such as auxins and cytokinins, to the culture medium; however, the optimal hormonal conditions for differentiation vary depending on plant species, genotypes, and tissue types.Therefore, establishment of optimal hormonal conditions to induce differentiation is required for every explant type.To date, several genes have been found to affect cell differentiation, such as ISOPENTENYLTRANSFERASE (IPT) ( Akiyoshi et al., 1984;Barry et al., 1984), SHOOT MERISTEMLESS (STM) (Barton and Poethig, 1993), LEAFY C OT Y L ED O N1 ( LEC1) (Lotan et al.,1 998 ), LEAFY COTYLEDON2 (LEC2) (Stone et al., 2001), GROWTH-REGULATING FACTOR-GRF-INTERACTING FACTOR (GRF-GIF) (Debernardi et al., 2020), BABY BOOM (BBM) (Boutilier et al., 2002), and WUSCHEL (WUS) (Laux et al., 1996).Therefore, these genes are also called developmental regulators (DRs).
BBM encodes an AP2/ERF transcription factor (Boutilier et al., 2002), which functions as a key regulator of zygotic embryogenesis in Arabidopsis (Chen et al., 2022a).Somatic embryo formation from the tissues of regenerated transgenic plants where Brassica BBM was overexpressed has been reported in Arabidopsis, rapeseed, tobacco, and sweet pepper (Boutilier et al., 2002;Heidmann et al., 2011).Also, during cell culture, the introduction and overexpression of Brassica BBM in Chinese white poplar calli induced somatic embryos (Deng et al., 2009).
WUS encodes a homeobox transcription factor that mainly localizes to the shoot apical meristem (SAM) to maintain stem cell identity (Laux et al., 1996).WUS is also involved in embryogenesis, and its overexpression induces somatic embryogenesis in Arabidopsis (Zuo et al., 2002;Ikeda et al., 2009).In addition to its homeodomain, the C-terminal region of the WUS protein contains three transcriptional regulatory domains: an acidic domain, a WUSbox, and an ethylene-responsive element binding factor-associated amphiphilic repression (EAR-like) motif (Ikeda et al., 2009).
However, these previous studies using DRs have not completely omitted the application of PGRs to the tissue or cell culture media for plant regeneration.The main bottleneck in artificial plant cellular differentiation is the difficulty in establishing the appropriate culture conditions, especially PGR application recipe, for each explant material.To overcome the obstacles, a versatile methodology to regulate cellular differentiation using DRs would be useful for biotechnological breedings.In addition, detailed information on the gene expression profiles and physiology regulated by DRs during cell differentiation will be informative for further improvements in plant molecular science and biotechnological breeding.Several studies have reported that phytohormone signaling in regenerated transgenic plants is altered by the ectopic expression of DRs (Li et al., 2019;Chen et al., 2022b;Zhou et al., 2023).However, how DR expression affects endogenous gene expression and physiology in cells undergoing differentiation has not been understood completely.
In this study, we utilized Arabidopsis BBM and WUS and modified the expressions and proteins to achieve autonomous differentiation of the transgenic cells.Here, we report that Brassicaceae Arabidopsis BBM and WUS successfully induced autonomous dedifferentiation and redifferentiation of transgenic cells without the application of PGRs during the culture process in Solanaceae tobacco, petunia, and Asteraceae lettuce.Using the differentiating transgenic tobacco cells obtained with the above system, we performed phytohormone measurements and RNA-seq analyses.We herein provide information on the physiological and gene expression backgrounds that enable autonomous cell dedifferentiation.

Vector construction
The primers used for vector construction are listed in Supplementary Table 1.BBM (AT5G17430) coding region (CDS) was PCR-amplified using cDNA from A. thaliana pistils at 7 dayafter flowering and was then cloned into pCR® 8/GW/TOPO (Thermo Fisher Scientific, Inc., Waltham, M.A., USA).The BBM CDS was inserted into pGWB2 (Nakagawa et al., 2007) using the LR reaction (Thermo Fisher Scientific, Inc.).The PCR-amplified fragment of Ipomoea batatas Myb (IbMyb) expression cassette (35Sp:IbMyb:NosT), which induces anthocyanin biosynthesis in the transgenic tobacco cells for visible marker selection (Sato et al., 2023), was further inserted into PmeI-SbfI site using the In-Fusion® HD cloning kit (Takara Bio Inc., Shiga, Japan), producing a pGWB_BBM binary vector (Supplementary Figure 1B).The 35Sp:WBBMVP16:HSPT region was cloned into an entry vector (pUC_35Sp:WBBMVP16) and transferred to pGWB1 (Nakagawa et al., 2007) via the LR reaction.Then an IbMyb expression cassette was further inserted into PmeI-SbfI site by In-Fusion®, producing a pGWB_WBBMVP16 binary vector (Supplementary Figure 1C).The CDS of WUS (AT2G17950) was amplified using cDNA from A. thaliana flower buds by PCR.The amplicons of A. thaliana ribosomal protein subunit 5A (RPS5A) promoter (AT3G11940) and the WUS CDS were inserted into a NotI site on pUC_35Sp:WBBMVP16 entry vector through In-Fusion®.The constructed 35Sp:WBBMVP16:NosT_RPS5Ap:WUS:NosT region was amplified, then inserted into the BamHI-SalI site on pKI1.1Rvector (Tsutsui and Higashiyama, 2017) through In-Fusion®.Further, the IbMyb expression cassette was inserted into a PmeI site by In-Fusion®.The final binary vector was named pKI_WBBMVP16&WUS (Supplementary Figure 1G).PCRamplified fragments of RPS5A promoter, SRDXWUSm1:NosT, and 35S promoter were inserted into the HpaI-NruI site on pKI_WBBMVP16&WUS vector through In-Fusion®, producing a binary vector pKI_WBBMVP16&SRDXWUSm1 (Supplementary Figure 1J).pGWB_GFP (Supplementary Figure 1A) was obtained by LR reaction of pCR® 8/GW/TOPO_eGFP and pGWB2 (Nakagawa et al., 2007).The PCR-amplified 35Sp:BBM:NosT region of pGWB_BBM (Supplementary Figure 1B) was inserted i n t o t h e X m a I -P s i I s i t e s o f p K I _ W B B M V P 1 6 & W U S (Supplementary Figure 1G) through In-Fusion®, producing a binary vector pKI_BBM&WUS (Supplementary Figure 1F).The SmaI-PsiI region was removed from pKI_WBBMVP16&WUS (Supplementary Figure 1G), and then self-ligated using Ligation high Ver. 2 (TOYOBO Co. Ltd., Osaka, Japan) to produce the binary vector pKI_WUS (Supplementary Figure 1D).The SmaI-PsiI regions of pKI_WBBMVP16&SRDXWUSm1 (Supplementary Figure 1J) were removed and self-ligated to produce the binary vector pKI_SRDXWUSm1 (Supplementary Figure 1E).The WUS:NosT:35Sp region was inserted into the PmlI-NruI site on pKI_WBBMVP16&SRDXWUSm1 (Supplementary Figure 1J) through In-Fusion®, producing a binary vector pKI_WBBMVP16&SRDXWUS (Supplementary Figure 1I).The A p a I -P m l I s i t e s o f p K I _ W B B M V P 1 6 & S R D X W U S m 1 (Supplementary Figure 1J) was replaced with the HSPT:RPS5Ap: WUS (partial) fragment, producing pKI_WBBMVP16&WUSm1 (Supplementary Figure 1H).All the series of T-DNA schemes constructed for Agrobacterium-mediated gene transfer are shown in Supplementary Figure 1.BBM and WUS expression was controlled by the cauliflower mosaic virus 35S and A. thaliana RPS5A promoters, respectively.The expression and functions of BBM and WUS were examined.For BBM, the 5'-leader sequence of tobacco mosaic virus (translational enhancer W) (Gallie, 2002) was inserted upstream of the CDS, and a translational activator domain of VP16 from herpes simplex virus (Triezenberg et al., 1988) was fused to the C-terminal (WBBMVP16, Supplementary Figure 1C).For WUS, two amino acid mutations were inserted into the WUSbox (WUSm1) and an artificial strong repression domain, Superman Repression Domain X (SRDX) (Hiratsu et al., 2003), was fused to the N-terminal end of WUSm1 (Ikeda et al., 2009) (SRDXWUSm1, Supplementary Figure 1E).

Agrobacterium-mediated gene transfer
The constructed binary vectors were transferred into Agrobacterium tumefaciens strain EHA105.Agrobacterium tumefaciens containing each binary vector was grown in Lysogeny Broth (LB) liquid medium containing 20 mg l -1 rifampicin, 30 mg l -1 chloramphenicol, and an appropriate antibiotic for each binary vector (50 mg l -1 kanamycin for pGWB, or 100 mg l -1 spectinomycin for pKI) at 28°C overnight in a shaking incubator (120 rpm).The Agrobacterium solution was diluted with MS liquid medium (OD 600 = 0.1) containing 3% sucrose and 100 mM acetosyringone (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).In vitro-grown tobacco leaves were cut into leaf explants (5.5-mm diameter) using a cork borer (NONAKARIKAKI Co., Ltd, Tokyo, Japan).For infection, 10-20 leaf explants were soaked in Agrobacterium solution for 1 min, then co-cultured on MS medium containing 3% sucrose and 0.8% agar for 3 days in the dark.Afterward, the leaf explants were transferred to an MS selection medium containing 3% sucrose, 0.8% agar, 40 mg l -1 hygromycin B, and 25 mg l -1 meropenem (Sumitomo Dainippon Pharma Co., Ltd., Tokyo, Japan), and subcultured to a new MS selection medium every two weeks.Two and four weeks after Agrobacterium infection (2WAI and 4WAI), the cell differentiation phenotypes were evaluated by counting the number of calli and shoots originating from each leaf explant culture.Three independent experiments were conducted to evaluate each binary vector.
Agrobacterium-mediated transformations for lettuce and petunia were carried out similarly to tobacco with minor modifications.Leaf discs (5.0-mm diameter) were infected, and a half-strength MS medium was used for Agrobacterium inoculation and co-culture.After co-culture, the leaf discs were cultured without selection pressure of hygromycin for a few weeks, followed by subculturing to a new MS selection medium containing 10−40 mg l -1 hygromycin B, 25 mg l -1 meropenem, 3% sucrose, and 0.8% agar.Shoots that emerged from the leaf explants were detached and transferred to the MS selection medium.Each grown and rooted plant were maintained in the culture vessels with MS selection medium.

Genomic PCR analysis
Genomic DNA was isolated from tobacco calli and shoots originating from infected leaf explants.For petunia and lettuce, small leaves derived from the shoots showing hygromycin resistance were used for genomic PCR.The calli and shoots were homogenized in DNA extraction buffer (containing 20 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0), and 10% SDS) and then centrifuged.After a phenol/chloroform treatment and ethanol precipitation, the obtained pellets were dissolved in water.Primers for IbMyb, Arabidopsis BBM and WUS, and Virulence D (VirD, for pTiBo542) detection (Supplementary Table 1) were used for PCR.

Real-time quantitative RT-PCR (qRT-PCR)
Total RNA was isolated using ISOSPIN Plant RNA (NIPPON GENE Co., Ltd., Tokyo, Japan).The cDNA was synthesized from 200 ng of total RNA using ReverTra Ace® (TOYOBO Co., Ltd).qRT-PCR was performed using KOD SYBR qPCR Mix (TOYOBO Co., Ltd) and Applied Biosystems StepOnePlus Real Time PCR System (Thermo Fisher Scientific, Inc.), using Comparative C T (DDC T ) method.For all qRT-PCR analyses, N. tabacum Elongation Factor 1-alpha (EF1a, AF120093) was used as the reference gene (Schmidt and Delaney, 2010).The primers used in each qRT-PCR analysis were listed in Supplementary Table 1.All primers that amplify WUS specifically do not distinguish WUS, SRDXWUS, and SRDXWUSm1.

RT-PCR analysis
Independent tobacco calli and shoots originating from the infected leaf explants were sampled as putative transgenic individuals.Total RNA was isolated using ISOSPIN Plant RNA.The cDNA was synthesized from 200 ng of total RNA using ReverTra Ace®.RT-PCR was performed using the primers listed in Supplementary Table 1.

Sample preparation for phytohormonequantitative analysis and RNA-seq
Five groups were used for phytohormone-quantitative analysis and RNA-seq: non-infected leaf explants (control), leaf cell cultures at 2 and 4WAI with WBBMVP16&WUS (2w:WBBMVP16&WUS and 4w:WBBMVP16&WUS), and leaf cell cultures at 2 and 4WAI with WBBMVP16&SRDXWUSm1 (2w:WBBMVP16&SRDXWUSm1 and 4w:WBBMVP16&SRDXWUSm1) (Figure 1A).Each Agrobacterium infection was carried out three or four times as a biological replicate.Five to ten leaf explants were collected.
For phytohormone-quantitative analysis, the collected samples were ground in liquid nitrogen with mortar and pestle and kept at −80°C until the analysis (following section 3.8).For total RNA extraction, the samples were soaked in RNA Save (Cosmo Bio Co., Ltd., Tokyo, Japan) and kept at −80°C until the experiments (following section 3.9).

RNA-seq analysis
Total RNA was isolated from each sample using ISOSPIN Plant RNA.An RNA-seq library was prepared using the Lasy-Seq ver.1.1 protocol (Kamitani et al., 2019) using 120 ng of total RNA.Each library was sequenced in triplicates using the single-end mode of the Illumina HiSeqX platform (Illumina Inc., San Diego, C.A., USA).Library preparation and sequencing were performed by Clockmics Inc. (Osaka, Japan).
The raw RNA-seq reads were pre-processed in Trimmomatic ver.0.39 (Bolger et al., 2014) using default parameters, and the trimmed reads were aligned to the reference tobacco TN90 genome (Sierro et al., 2014) using HISAT2 ver.2.2.1 (Kim et al., 2015).Gene expression levels were quantified using StringTie ver.2.2.1 (Pertea et al., 2015).Principal component analysis (PCA) was performed using the graphical user interface for the R package TCC (TCC-GUI ver.1.0 pipeline) (Su et al., 2019) using the expression data.Differentially expressed genes (DEGs) between the two groups were detected using the TCC-GUI pipeline with a false discovery rate (FDR) of 0.05, and between the three groups using the method of combining the R packages baySeq ver.2.24.0 and TCC ver.1.30.0 (Osabe et al., 2019) with FDR of 0.05.The expression levels of the DEGs were visualized using the R package heatmap3 ver.1.1.9(Zhao et al., 2014) using read count data normalized by the R package TCC.Gene Ontology (GO) enrichment analysis of the DEGs was performed using the R package clusterProfiler ver.3.18.1 (Yu et al., 2012).GO term networks were also visualized using the same R package.DEGs were annotated from the NCBI database (https://www.ncbi.nlm.nih.gov/).Orthologous genes of the DEGs in A. thaliana were identified using OrthoDB v11 (https:// www.orthodb.org/),and annotations of the orthologous genes were obtained from the TAIR database (https://www.arabidopsis.org/).Commonly upregulated DEGs in two-and three-group comparisons were visualized using the R package VennDiagram ver.1.7.1 (Chen and Boutros, 2011).The raw RNA-seq data was deposited at the NCBI Sequence Read Archive (https:// www.ncbi.nlm.nih.gov) with an accession number PRJNA1049661.

Effects of Arabidopsis BBM and WUS genes introduced into tobacco, petunia, and lettuce leaf cells
First, we tested the gene expression scheme using Arabidopsis BBM and WUS as DRs, which enabled autonomous cell differentiation of transgenic tobacco cells in Agrobacteriuminfected leaf explants during the culture process.
Leaf explants from the experimental controls, non-infected explants, and explants infected with pGWB_35Sp:GFP, did not show any reaction to cell differentiation, and were entirely browned by 4WAI (Supplementary Figure 1A).The results indicated that tobacco leaf cells could not differentiate or survive on a medium without PGRs, and the stress of Agrobacterium infection and gene transfer did not have different effects.
The BBM and WBBMVP16-introduced leaf cells also died by 4WAI (Supplementary Figures 1B, C).A previous study reported that overexpression of SRDXWUSm1 enhanced adventitious shoot formation in Arabidopsis (Ikeda et al., 2020).However, WUS and SRDXWUSm1-introduced leaf cells did not survive (Supplementary Figures 1D, E), indicating that the introduction of Arabidopsis BBM or WUS alone was insufficient to induce tobacco cell differentiation.Next, the combination of BBM and WUS was examined.Although the introduction of BBM&WUS was insufficient (Supplementary Figure 1F), WBBMVP16&WUS-introduced leaf cells differentiated into calli after 4WAI (Figure 1A; Supplementary Figure 1G).Furthermore, WBBMVP16&SRDXWUSm1-introduced leaf cells showed a more accelerated differentiation phenotype after 4WAI (Figure 1A; Supplementary Figure 1J).Unlike the WBBMVP16&WUS-introduced leaf cells, WBBMVP16& SRDXWUSm1-introduced leaf cells differentiated into greenish, organ-like structures, in addition to small calli at 2WAI (Figure 1A; Supplementary Figure 1J).As of 4WAI, the calli proliferated vigorously, and a few adventitious shoots emerged from WBBMVP16&SRDXWUSm1-introduced leaf cells.Significantly higher numbers of calli and shoots were obtained from leaf explant cultures introduced with WBBMVP16& SRDXWUSm1 (Figure 1B; Supplementary Figure 2).To investigate whether amino acid mutations in WUS-box (WUSm1) or SRDX fusion to the N-terminal of WUS (SRDXWUS) was involved in the accelerated differentiation phenotype of WBBMVP16&SRDXWUSm1-introduced leaf cells, WBB MVP16&WUSm1 (Supplementary Figure 1H) and WBBM VP16&SRDXWUS (Supplementary Figure 1I) were evaluated.While WBBMVP16&WUSm1-introduced leaf cells did not induce any cell differentiation and died by 4WAI (Supplementary Figure 1H), WBBMVP16&SRDXWUS-introduced leaf cells differentiated calli and shoots vigorously (Supplementary Figure 1I).Compared to WBBMVP16&SRDXWUS, the number of calli that emerged from a leaf explant culture was higher in WBBMVP16&SRDXWUSm1, whereas the number of shoots did not show significant differences between the two constructs (Supplementary Figure 2).In the present study, introducing Arabidopsis WBBMVP16&WUS, WBBMVP16&SRDXWUS, and WBBMVP16&SRDXWUSm1 succeeded in the autonomous differentiation of tobacco leaf cells without applying PGRs during the tissue culture process.
To confirm the observed phenotypes (Figure 1; Supplementary Figure 1) were linked to expression of the introduced genes, real-time qRT-PCR was performed for tobacco leaf explants introduced the WBBMVP16&WUS and WBBMVP16&SRDXWUSm1 (Supplementary Figure 3).As a result, the transcription of Arabidopsis BBM and WUS was confirmed in the infected leaf explants during the culture by 4WAI.Compared to WUS driven by RPS5A promoter, BBM driven by 35S promoter showed significantly higher expression levels in all the 2 and 4WAI leaf explants.
We further evaluated whether the autonomously differentiated calli and shoots were the transgenics expressing BBM and WUS.Genomic PCR analysis was performed using calli and shoots originated from WBBMVP16&WUSand WBBMVP16& SRDXWUSm1-introduced leaf cells.As a result, the Arabidopsis BBM and WUS were detected in the callus and shoot lines obtained with WBBMVP16&WUS and WBBMVP16&SRDXWUSm1, but the VirD gene from Agrobacterium was not, showing that these were transgenic (Supplementary Figure 4).IbMyb, which has been introduced as a visible marker of anthocyanin pigmentation (Kim et al., 2010;Sato et al., 2023), was also detected; however, anthocyanin pigmentation was hardly observed in the differentiated cells (Figure 1A; Supplementary Figures 1G, I, J).To further verify that each transgene was transcribed in transgenic calli and shoots, RT-PCR was performed.As a result, all the IbMyb, Arabidopsis BBM, and WUS expressions were detected (Supplementary Figure 5).Thus, the downstream anthocyanin biosynthesis pathway induced by IbMyb may have been hampered in cells expressing BBM and WUS.
Furthermore, we tested the effect of these expression constructs with different plant species, petunia and lettuce, which are receptive to Agrobacterium-mediated gene transfer.As a result, autonomous differentiation of calli and shoots from leaf explants cultured on the PGR-free medium was observed at 4WAI (Supplementary Figure 6).The presence of transgenes in the obtained hygromycin-resistant shoots was confirmed (Supplementary Figure 7).
The ectopic overexpression of DRs has been reported to cause morphogenic abnormalities in transgenic plants (Boutilier et al., 2002;Srinivasan et al., 2007;Chen et al., 2022b), and the higher expression level linked to severe phenotype in tobacco (Srinivasan et al., 2007).The obtained tobacco transgenic plants in the present study showed malformation with various intensities.The qRT-PCR of BBM and WUS in the plant lines showing 'nearly normal', 'moderate,' and 'severe' malformations showed similar co-relation between DRs levels and morphologies (Supplementary Figure 8).

Phytohormone contents in differentiating tobacco leaf cells
Cultured cells differentiating without external PGR treatment are suitable for analyses to obtain pure endogenous cellular behaviors.Phytohormones are important regulators of cell division and differentiation.Because the differentiation of leaf cells caused by the introduction of WBBMVP16&WUS and WBBMVP16&SRDXWUSm1 showed dramatically different patterns (Figure 1A; Supplementary Figures 1G, J), the underlying hormonal behaviors were analyzed.Six phytohormones (auxins, cytokinins, GAs, ABA, SA, and JA) and their metabolites in the leaf explant cultures at 2 and 4WAI were measured (Figure 2; Supplementary Table 2).
The active auxin indole-3-acetic acid (IAA) and the irreversible catabolite indole-3-acetyl-L-aspartic acid (IAAsp) showed similar behaviors in the WBBMVP16&WUSand WBBMVP16 &SRDXWUSm1-introduced leaf cultures by 4WAI.At 2WAI, compared with non-infected (0-week cultured) leaf explants, the IAA content in WBBMVP16&SRDXWUSm1 was significantly higher, whereas it was significantly lower in WBBMVP16&WUS.The IAA content was further elevated to a similar level by 4WAI in both WBBMVP16&WUSand WBBMVP16&SRDXWUSm1-introduced leaf cultures.The IAAsp content in WBBMVP16&SRDXWUSm1introduced leaf cultures was also higher than that in WBBMVP16&WUS-introduced leaf cultures, especially at 2WAI.
In comparison with the non-infected leaf explants, similar cytokinin content, except for dihydrozeatin (DZ), was observed in both WBBMVP16&WUS and WBBMVP16&SRDXWUSm1introduced leaf cultures.At 2WAI, N6-(D2-isopentenyl) adenine (iP) content was yet negligible in WBBMVP16&WUS, whereas it was significantly higher in WBBMVP16&SRDXWUSm1.The iP Autonomous differentiation of tobacco leaf cells with introduced Arabidopsis BABY BOOM (BBM) and WUSCHEL (WUS) genes.(A) The phenotypes of tobacco leaf explant cultures with introduced WBBMVP16&WUS (upper photos) and WBBMVP16&SRDXWUSm1 (lower photos) via Agrobacterium infection.Photos at two weeks (2w) and four weeks (4w) after Agrobacterium infection are shown, respectively.The left image is an explant used for infection (F 5.5 mm).A differentiated shoot from the leaf explant cells with introduced WBBMVP16&SRDXWUSm1 is marked with a white arrow.White triangles represent anthocyanin pigmentation rarely observed in calli.Bars = 5 mm.(B) The number of calli and shoots that emerged from a leaf explant 4 weeks after the infection.Three biological replicates of Agrobacterium infection were carried out, and the data is indicated together.Red, green, and blue dot-plots represent the number of calli (left plots) and shoots (right plots) that originated from a non-infected (control) leaf explant or one with introduced WBBMVP16&WUS and WBBMVP16&SRDXWUSm1, respectively.Each violet diamond represents the mean.Asterisks indicate significant differences (p < 0.01) between WBBMVP16&WUS and WBBMVP16&SRDXWUSm1, detected by Welch's t-test.
elevations in both WBBMVP16&WUSand WBBMVP 6&SRDXWUSm1-introduced leaf cultures continued for 4WAI, and the overall contents in the WBBMVP16&SRDXWUSm1introduced leaf cultures were significantly higher than those in WBBMVP16&WUS.An earlier elevation of trans-zeatin (tZ) was observed in WBBMVP16&SRDXWUSm1 than in WBBMVP 16&WUS, and the content level was maintained by 4WAI.Although tZ was negligible in the WBBMVP16&WUS-introduced leaf cultures at 2WAI, it reached a level similar to that of WBBMVP16&SRDXWUSm1 at 4WAI.Elevated cis-zeatin (cZ) level was detected in WBBMVP16&SRDXWUSm1 compared to the non-infected leaf cultures at 2WAI, and each level was maintained until 4WAI.cZ content at 4WAI was significantly higher in WBBMVP16&WUS than in WBBMVP16&SRDXWUSm1.
The content behaviors were various depending on the GA type.One of the active GAs, GA 1 , was maintained at negligible levels by 4WAI in both WBBMVP16& WUS-and W BBMVP16& SRDXWUSm1-introduced leaf cultures.Significantly higher levels of GA 19 , GA 24 , and GA 53 were detected in the WBBMVP16& SRDXWUSm1-introduced leaf cultures at 4WAI than in those of WBBMVP16&WUS, whereas continuous elevation of GA 24 was also observed in the WBBMVP16&WUS-introduced leaf cultures.The content of GA 4 , an active GA, was below the detection level in all samples analyzed (Supplementary Table 2).
Compared to the non-infected leaf explants, the ABA content was lower in the WBBMVP16&WUS-introduced leaf cultures throughout the four weeks of culture from infection.In contrast, the ABA level was elevated in the WBBMVP16&SRDXWUSm1introduced leaf cultures at the 4WAI, showing overall higher levels than that of WBBMVP16&WUS.
No significant differences in SA, JA, and JA-Ile content were detected throughout the four weeks of culture in the comparison of WBBMVP16&WUSand WBBMVP16&SRDXWUSm1-introduced leaf cultures.Statistically, the JA-Ile content at 2WAI was Phytohormone content in differentiating leaf explant cells with introduced WBBMVP16&WUS and WBBMVP16&SRDXWUSm1.The phytohormone content in the leaf explant cultures was measured at two and four weeks after Agrobacterium infection.The y-axis represents the concentration (pmol/g fresh weight (FW)) and the x-axis represents the culture period (0, 2, and 4 weeks (w)).Orange and blue dots represent the values of WBBMVP16&WUSand WBBMVP16&SRDXWUSm1-introduced leaf explant cultures, respectively.Non-infected leaf explants were analyzed as 0-week cultured samples (0w), and the results are indicated by gray dots.Light green crosses and vertical black bars associated with boxplots represent the mean and standard deviation, respectively (n = 4).The horizontal black bar in each boxplot represents the median value.IAA, Indole-3-acetic acid; IAAsp, indole-3-acetyl-L-aspartic acid; iP, N6-(D2-isopentenyl) adenine; tZ, trans-Zeatin; DZ, dihydrozeatin; cZ, cis-Zeatin; GA, Gibberellin; ABA, Abscisic acid; SA, Salicylic acid; JA, Jasmonic acid; JAIle, Jasmonic acid with isoleucine.Asterisks indicate significant differences (*p < 0.05, **p < 0.01) between WBBMVP16&WUSand WBBMVP16&SRDXWUSm1-introduced leaf explant cultures during the same culture period, as detected by Welch's t-test.Different lowercase letters in the same color indicate significant differences (p < 0.05) among the three time points (0, 2, and 4w), as detected using the Tukey-Kramer's test.ND, not detected (below the quantification limit).All measurement data are shown in Supplementary significantly lower in the WBBMVP16&SRDXWUSm1-introduced leaf cultures.Focusing on active auxin (IAA) and cytokinins (iP, tZ), each phytohormone level tended to increase earlier and was higher in the leaf explant cultures at 2WAI with WBBMVP 16&SRDXWUSm1-introduced leaf cultures than in those with WBBMVP16&WUS.

Gene expression profiles in differentiating tobacco leaf cells
To analyze transcriptional behavior during cell differentiation, we performed RNA-seq of tobacco leaf cells cultured for 2 or 4WAI with WBBMVP16&WUS and WBBMVP16&SRDXWUSm1, and 0week cultured leaf explants without Agrobacterium infection (control).PCA showed that the three biological replicates of each sample represented by the same color clustered together (Figure 3).Notably, the groups of 2w:WBBMVP16&SRDXWUSm1 and 4w:WBBMVP16&WUS were located close together.
In summary, GO terms involved in phytohormones were enriched with genes upregulated by introducing WBBMVP16&SRDXWUSm1 at 2 and 4WAI, compared to WBB MVP16&WUS.The GO terms related to phytohormones differed at each time point; GO terms related to auxin and cytokinin were enriched at 2WAI, whereas those related to auxin and ABA were enriched at 4WAI.These GO terms were associated with signaling or transport but not with biosynthesis and metabolism.
The DEGs detected in this study were summarized as Venn diagram (Supplementary Figure 15); significantly upregulated in WBBMVP16&SRDXWUSm1-introduced leaf cells at both 2 (Figure 4) and 4WAI (Figure 5), and gradually upregulated during the culture process in WBBMVP16&SRDXWUSm1-introduced leaf cells (Figure 6).Among 19 genes commonly detected as significantly upregulated in WBBMVP16&SRDXWUSm1-introduced leaf cells at both 2 (blue) and 4WAI (green in Supplementary Figure 15), cell proliferation and auxin-related GO terms were included (FDR < 0.05) (Supplementary Table 7).In this analysis, one common gene LOC107801243 was detected in the three-group comparison (purple in Supplementary Figure 15; Supplementary Table 7).On the other hand, no significant GO terms were detected for the intersect DEGs that were downregulated (data not shown).

qRT-PCR of the genes detected as DEGs in WBBMVP16&SRDXWUSm1introduced leaf cell cultures
To evaluate the actual expression behaviors of DEGs affected by WBBMVP16&SRDXWUSm1 introduction, qRT-PCR was performed for four genes: LOC107773826 down-regulated over time (Supplementary Table 5), LOC107782639 and LOC107795218 showed higher levels compared to WBBMVP16&WUS (Supplementary Table 7), and LOC107801243 upregulated over time and higher compared to WBBMVP16&WUS (Supplementary Table 7).LOC107773826 and LOC107782639 are the putative orthologs of Arabidopsis TCP3/4/10 and IAA26, respectively (Supplementary Tables 3-5).The LOC107795218 is annotated as a putative phytosulfokine-3 gene (XM_016617810).The LOC107801243 is identified as a putative paralogue gene of NAD + -dependent protein deacetylase HST1 (XP_016480848), as detected by tBLASTn search (NCBI).Expression analysis revealed that LOC107773826 (TCP3/4/10) was consistently downregulated in all infected leaf cell cultures at 2 and 4WAI compared to the control (non-infected leaf explant).Notably, expression levels were lower in leaf cells introduced with the WBBMVP16&SRDXWUSm1 than in those with WBBMVP16&WUS (Figure 7A).Both LOC107782639 (IAA26) and LOC107795218 (Phytosulfokine-3) showed higher expression levels in WBBMVP16&SRDXWUSm1-introduced leaf cells than in WBBMVP16&WUS-introduced cells at 2 and 4 WAI (Figure 7A).LOC107801243 (HST1) showed high expression in the WBBMVP16&SRDXWUSm1-introduced leaf cell cultures, further upregulated at 4WAI (Figure 7A).These qRT-PCR results corresponded with DEGs identified from RNA-seq analysis.Furthermore, qRT-PCR of the same four genes was performed for transgenic calli and shoots derived from leaf explant cultures.Compared to the non-infected leaf explants (control), LOC107773826 (TCP3/4/10) expression was downregulated in all tested calli and shoots (Figure 7B).In general, expression levels of LOC107782639 (IAA26), LOC107795218 (Phytosulfokine-3), and LOC107801243 (HST1) were higher inWBBMVP16&SRDXWUSm1 samples compared to WBBMVP16&WUS, particularly in callus (Figure 7B).In the present study, we demonstrated that the combined expression of Arabidopsis BBM and WUS in transgenic tobacco, petunia, and lettuce leaf cells resulted in autonomous cell differentiation without the application of PGRs during culture process (Figure 1A; Supplementary Figures 1-7).Single and combined introduction of BBM and WUS, which are regulated by the 35S and RPS5A promoters, respectively, was insufficient to induce cell differentiation (Supplementary Figures 1B, D, F).A single introduction of WBBMVP16, in which the translation enhancer omega sequence and the VP16 transcriptional activator were applied, was still insufficient (Supplementary Figure 1C).In addition, a single introduction of SRDXWUSm1, in where two amino acid mutations were inserted into the WUS-box and an artificial strong repression domain, SRDX, was fused to the Nterminal end of WUSm1, did not induce cell differentiation (Supplementary Figure 1E).In contrast, the combined expression of WBBMVP16 and WUS (WBBMVP16&WUS) successfully Relative expression levels of four genes detected as DEGs by WBBMVP16&SRDXWUSm1 introduction.(A) q-RT PCR analysis with samples derived from three biological replicates of infection with WBBMVP16&WUS and WBBMVP16&SRDXWUSm1.The x-axis represents the 2 or 4-week-cultured leaf explant cultures (2w and 4w) introduced with each construct.(B) qRT-PCR with transgenic calli and shoots samples.The shoots introduced with WBBMVP16&WUS were not analyzed because of the scarce formation (Figure 1B).Three independent samples detached from the differentiating leaf explant cell culture were analyzed.The y-axis in all graphs represents the relative quantification (RQ; 2 -DDCt ) compared to the control (non-infected leaf explant).The error bars represent the maximum and minimum RQ values of three reaction replicates (95.0%confidence level).
induced transgenic calli (Figure 1A; Supplementary Figure 1G).In a previous study, Arabidopsis and Brassica napus BBM were evaluated in tobacco, and inducible differentiation from leaf explant cells was achieved by the supportive application of cytokinins, and leaves from the 35S::BBM transgenic tobacco plants did not cause cellular differentiation in PGR-free culture conditions (Srinivasan et al., 2007).Taken together with the previous insights and our results, the single expression of translationally and functionally enhanced BBM could alter the cell physiology involved in endogenous phytohormone for differentiation; however, it might still be insufficient to cause morphological alterations as apparent differentiation.As for WUS, the expression of Arabidopsis WUS induces adventitious shoot formation from the root tissues in Arabidopsis and tobacco (Rashid et al., 2007;Ikeda et al., 2009Ikeda et al., , 2020)).Therefore, to induce adventitious shoot differentiation from somatic cells by the ectopic expression of WUS, it may be critical that somatic cells have a root-pertaining genetic background.Thus, the differentiation achieved by the combined expression of WBBMVP16 and WUS (or SRDXWUS, SRDXWUSm1) suggests that these DRs synergistically influence gene expression and physiology, resulting in cellular differentiation.In leaf cell cultures exhibiting autonomous differentiation, relatively lower levels of WUS expression were observed compared to BBM expression levels (Supplementary Figure 3).This observation supports the speculation that WUS triggers cell proliferation and organogenesis with substantial physiological modifications by BBM.The malformation patterns observed in the regenerated transgenic plants were associated with higher DR expression levels, particularly a significant BBM level (Supplementary Figure 8).To regulate the morphology of regenerated plants, employing inducible promoters would improve the current system.
The combined expression of WBBMVP16 and SRDXWUSm1 (or SRDXWUS) showed a different differentiation pattern compared to WBBMVP16&WUS, showing faster and more accelerated shoot differentiation in tobacco transgenic cells (Figure 1A; Supplementary Figures 1I, J).A similar tendency was observed in the case of petunia and lettuce (Supplementary Figure 6).The ectopic expression of SRDXWUSm1 is more effective than WUS in inducing adventitious shoot formation from somatic cells in Arabidopsis (Ikeda et al., 2020).Similarly, our study demonstrated a significant effect on autonomous shoot organogenesis in tobacco cells through the expression of SRDXWUS or SRDXWUSm1 in combination with WBBMVP16, indicating that SRDX fusion to the N-terminal of WUS was effective for the cellular differentiation also in tobacco cell (Supplementary Figures 1, 2).Regarding the WUSbox mutation, WUSm1 showed a reduced ability for autonomous callus formation compared to WUS (Supplementary Figures 1G, H,  2).In contrast, SRDXWUSm1 induced higher callus formation than SRDXWUS (Supplementary Figure 2).WUS function as a repressor in maintaining pluripotent stem cells, and WUS-box is crucial for this role (Ikeda et al., 2009).It has been suggested that the fusion of the artificial strong repression domain SRDX to the WUSm1 generates a protein with enhanced repression activity, thereby promoting organogenesis through cytokinin signaling (Ikeda et al., 2020).Alternatively, it could be postulated that the acceleration of stem cell maintenance repression disruption by the WUS-box mutation was facilitated by SRDX fusion, thereby stimulating cell division in tobacco cells.

Phytohormone behaviors underlining dedifferentiation and redifferentiation
Because the present BBM and WUS expression system enabled autonomous cell differentiation on a PGR-free medium, pure endogenous phytohormone behaviors underlying cellular differentiation could be analyzed.To understand the swiftness and differences in cell differentiation observed in this study, phytohormone-quantitative analysis was performed (Figure 2; Supplementary Table 2).Active auxin (IAA) and cytokinin (iP and tZ) levels were higher in 2w:WBBMVP16&SRDXWUSm1 and 4w:WBBMVP16&WUS, in which callus induction was observed.The timing of the increase in active auxin/cytokinin content coincided with callus induction in tobacco leaf explants (Figure 1A).This result is consistent with the acknowledged theory that increased auxin and cytokinin levels in tissue culture medium promote dedifferentiation (Skoog and Miller, 1957;Phillips and Garda, 2019).
Focusing on 4WAI, the calli that emerged from WBBMVP16&SRDXWUSm1 induced a more shoot-like structure, indicating that redifferentiation was promoted compared to WBBMVP16&WUS.The contents of GA 19 , GA 24 , and GA 53 , which are precursors of active GA 1 or GA 4 (He et al., 2020), were significantly higher in WBBMVP16&SRDXWUSm1 than in WBBMVP16&WUS, whereas all the active GAs (GA 1 , GA 3, GA 4 , and GA 7 ) were not present at the quantitative level in any of the analyzed samples (Figure 2; Supplementary Table 2).Active GAs negatively regulate shoot regeneration in Arabidopsis (Ezura and Harberd, 1995).Active GAs and cytokinins act antagonistically during shoot regeneration in tobacco (Engelke et al., 1973).In our study, the accumulation of active GA precursors (Figure 2) suggests that active GAs synthesis was suppressed and endogenous cytokinins were not antagonized for shoot regeneration in WBBMVP16&SRDXWUSm1-introduced leaf cells.
In the present study, a significant increase in ABA was observed in 4w:WBBMVP16&SRDXWUSm1.Although the substantial contribution of ABA to cellular differentiation has not yet been elucidated (Shin et al., 2020), ABA is known to crosstalk with other phytohormones, and is involved in plant growth regulation and stress (Skubacz et al., 2016).Thus, the observed elevation suggests the involvement of ABA in organogenesis in the coordinated balance between auxin and cytokinin that maintained this level.

Gene expression differences between the differentiated cells caused by WBBMVP16&WUS and WBBMVP16&SRDXWUSm1
In RNA-seq analysis, GO terms such as 'auxin-activated signaling pathways' and 'response to cytokinin' were enriched with the DEGs upregulated in 2w:WBBMVP16&SRDXWUSm1 (Figures 4B, C; Supplementary Figure 9).These GO terms included the putative orthologs of AUX/IAAs and PIN1/3/4/7 (Supplementary Table 3), which coincided with increased levels of active auxin (Figure 2).The other detected ortholog in the GO terms, Arabidopsis HP6, has been known to act as an inhibitor of cytokinin signaling (Mähönen et al., 2006;Müller and Sheen, 2007;Besnard et al., 2014).Moreover, it has also been reported that overexpression of HP6 in SAM suppresses type-A ARRs, which repress cytokinin signaling (Besnard et al., 2014).In our study, the tobacco orthologous gene of Arabidopsis HP6 was upregulated in 2w:WBBMVP16&SRDXWUSm1 more than in 2w:WBBMVP16&WUS.The calli induced by WBBMVP16&SRDXWUSm1 had greenish organ-like structures (Figure 1A; Supplementary Figure 1J).Therefore, it was suggested that upregulation of HP6 promoted the suppression of type-A ARRs, resulting in the activation of cytokinin signaling, cell division, and near-organogenic differentiation.Active cytokinin content was also higher in 2w:WBBMVP16&SRDXWUSm1 than in 2w:WBBMVP16&WUS (Figure 2), which is consistent with this hypothesis.Previous studies have reported that HP6 is directly activated by auxin (Bishopp et al., 2011;Besnard et al., 2014) and affects the localization of PIN1 in Arabidopsis (Moreira et al., 2013).Such interactions of HP6 with auxin and PINs may be involved in the differences in the differentiation phenotypes caused by WBBMVP16&WUS and WBBMVP16&SRDXWUSm1.In addition, GO terms related to the cell wall, especially the 'polysaccharide catabolic process' were significantly upregulated in 2w:WBBMVP16&SRDXWUSm1 compared to 2w:WBBMVP16&WUS (Figures 4B, C; Supplementary Table 3).A previous study reported that WUS homeobox-containing 13 (WOX13) is a key regulator to promote callus formation by modifying the cell wall properties in Arabidopsis (Ikeuchi et al., 2022).WOX13 directly upregulates cell wall-related genes involved in the polysaccharide catabolic process (Ikeuchi et al., 2022).WOX13 conserves the WUS-homeodomain and an acidic domain but lacks the WUS-box (Dolzblasz et al., 2016) and targets sequences that also target WUS (Ma et al., 2019;Ikeuchi et al., 2022).The promoted callus formation by SRDXWUSm1 observed in our study (Supplementary Figure 2) could reflect a similar activation to WOX13.
The 4w:WBBMVP16&SRDXWUSm1 leaf cells differentiated into calli and shoots, in contrast to 4w:WBBMVP16&WUS, in which only callus formation was observed (Figure 1A; Supplementary Figures 1G, J).The result that the active cytokinin iP content was higher (Figure 2) was reminiscent of the differentiation tendency of N. tabacum in tissue culture, where a higher ratio of cytokinin to auxin induces organogenesis (Skoog and Miller, 1957;Phillips and Garda, 2019).Although the GO terms related to cytokinin were not enriched, GO terms related to auxin and ABA were detected in the DEG upregulated in 4w:WBBMVP16&SRDXWUSm1 (Figure 5B; Supplementary Figures 10A, 11; Supplementary Table 4).In addition to auxin transport, response, and signaling, GO terms related to auxins included accessions involved in shoot development (Figure 5B;Supplementary Figures 10A,11;Supplementary Table 4).This result may reflect the adventitious shoot formation observed in the present study (Figure 1A; Supplementary Figure 1J).Although the role of ABA in plant regeneration has not been extensively studied, several studies have suggested its effects on shoot regeneration (Shin et al., 2020).For example, the leucine-rich repeat receptor-like kinase RECEPTOR-LIKE PROTEIN KINASE1 (RPK1), which is involved in ABA signaling, promotes shoot regeneration in Arabidopsis (Motte et al., 2014).As mentioned above, ABA is also known to crosstalk with other phytohormones (Skubacz et al., 2016), as reported that ABAinduced ABI5 inhibited PINs accumulation and auxin activity, for instance (Yuan et al., 2014).The fact that many GO terms related to stress were detected (Figure 5B; Supplementary Figure 10A) implies the involvement of ABA.In our study, the putative orthologs of ABI5 and PINs were detected as DEG that were upregulated in 4w:WBBMVP16&SRDXWUSm1 (Supplementary Table 4), suggesting that phytohormone crosstalk, especially by auxin and ABA, was influenced by organogenesis, such as shoot formation.
The primary GO terms detected in DEGs downregulated in 4w:WBBMVP16&SRDXWUSm1 were related to metabolism, which is likely involved in transcription and translation (Figure 5C; Supplementary Figure 10B).This result reflects the dominance of the above metabolic pathways for active cell division in callus formation (4w:WBBMVP16&WUS) rather than for organogenesis (4w:WBBMVP16&SRDXWUSm1).

Changes in gene expression over time in WBBMVP16&SRDXWUSm1-introduced leaf cells
No significant changes in the expression of genes related to phytohormones or regeneration were observed over time in WBBMVP16&WUS-introduced leaf cells, whereas GO terms related to the regulation of plant organogenesis were significantly downregulated over time in WBBMVP16&SRDXWUSm1introduced leaf cells.Genes related to the regulation of plant organogenesis were identified as the TCP3/4/10 orthologs (Supplementary Table 5), and qRT-PCR also showed the significant downregulation in WBBMVP16&SRDXWUSm1-introduced leaf cells and differentiated calli and shoots (Figure 7).TCP4 is involved in multiple plant developmental processes, such as leaf and flower morphogenesis, secondary cell wall biosynthesis, senescence, and hormone signaling in Arabidopsis (Palatnik et al., 2003;Schommer et al., 2008;Nag et al., 2009;Sarvepalli and Nath, 2011;Li et al., 2012;Sun et al., 2017).A previous study reported promotion of shoot regeneration in tcp3/4/10 mutant in Arabidopsis, indicating that TCPs are strong negative regulators of de novo shoot regeneration (Yang et al., 2020).The downregulation of TCP3/4/10 over time in WBBMVP16&SRDXWUSm1-introduced leaf cell cultures might account for the enhanced differentiation, while the significant difference in the expression level between differentiated calli and shoots was not observed in our study (Figure 7B).In addition, GO terms related to glutamine and asparagine were gradually downregulated in WBBMVP16&SRDXWUSm1-introduced leaf cells (Figure 6B; Supplementary Figure 13), and the ortholog genes of GDHs, GS2, and ASN1 were annotated (Supplementary Table 5).These genes are upregulated in the dark to activate nitrogen assimilation (Yoneyama and Suzuki, 2020;Liu et al., 2022).In the present study, chloroplast differentiation was observed in WBBMVP16&SRDXWUSm1-introduced leaf cells (Figure 1A; Supplementary Figure 1J), and all culture processes were performed under continuous fluorescent light.These results and the environmental conditions may account for the downregulation of these genes.In addition, GO terms related to stimuli were detected from five DEGs, including OSM34 and MLOs orthologs, which have been reported to be involved in the ABA response (Lim and Lee, 2014;Park and Kim, 2021).The relationship between the elevated ABA level at 4WAI compared to that at 2WAI (Figure 2) and the downregulation of these ortholog genes cannot be explained at present, although it could imply that altered ABA signaling influenced cellular differentiation.
The present study identified 19 genes as the common intersections among DEGs upregulated during culture over time by WBBMVP16&SRDXWUSm1 compared to WBBMVP16&WUS.Among these genes, LOC107801243 was upregulated compared to both the control and WBBMVP16&WUS (Supplementary Figure 15; Supplementary Table 7).The qRT-PCR analysis of selected genes LOC107782639 (IAA26), LOC107795218 (Phytosulfokine-3), and LOC107801243 (NAD + -dependent protein deacetylase HST1) showed similar behavior to those detected by RNA-seq, showing upregulation in both differentiated calli and shoots (Figure 7).The IAA26 belongs to the Aux/IAA transcription factor family and is known to respond to auxin.Therefore, the increased levels of auxins (Figure 2) may have contributed to the upregulation of LOC107782639.Phytosulfokine is a plant peptide growth factor that affects plant growth and differentiation (Ding et al., 2022).A study has shown that altered expression of an Arabidopsis phytosulfokine receptor influenced the callus formation capacity in response to PGRs (Matsubayashi et al., 2006).Considering the higher expression of LOC107795218 (Phytosulfokine-3) was observed in calli induced with WBBMVP16&SRDXWUSm1 (Figure 7B), enhanced LOC107795218 expression may be associated with callus induction.In the yeast Saccharomyces cerevisiae, it has been reported that NAD + -dependent protein deacetylase HST1 is a sensor of cellular NAD + levels involved in homeostasis (Bedalov et al., 2003).NAD + is converted from nicotinic acid imported from the culture medium.Additionally, yeast HST1 represses the expression of thiamin biosynthesis genes by binding to their promoters, thereby regulating thiamin homeostasis (Li et al., 2010).Nicotinic acid and thiamin are components of the MS medium.These findings and our results (Figure 7; Supplementary Figure 15; Supplementary Table 7) suggest that LOC107801243 was highly upregulated in actively differentiating cells importing extracellular vitamins.In our study, no orthologs of LEC1, LEC2, ABSCISIC ACIDINSENSITIVE3 (ABI3), and FUSCA3 (FUS3), which are known to be activated by BBM (Horstman et al., 2017), were detected.The present study examined DEGs resulting from the combined expression of SRDXWUS or SRDXWUSm1 with WBBMVP16.It is possible that these combinations could have influenced gene networks differently compared to the single expression of BBM alone.Future functional analysis of found DEGs and investigation of gene networks related to known DRs would provide further insights into cellular differentiation and organogenesis.

FIGURE 3
FIGURE 3Principal component analysis of gene expression levels in tobacco leaf explant cultures.The 'control' is the experimental control (non-infected leaf explant).Plots with the same color indicate biological replicates.2w and 4w, 2 (or 4) weeks after Agrobacterium infection.