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. 2024 Jul 24;30(8):1253–1263. doi: 10.1007/s12298-024-01493-y

A point mutation in the IAA14 promoter enhances callus formation and regeneration

Huifen Cao 1, Xiao Zhang 2,✉, Feng Li 1, Zhiping Han 1, Baopeng Ding 2
PMCID: PMC11341521  PMID: 39184564

Abstract

Callus formation induced by auxin accumulation is considered the first step of in vitro plant regeneration. In Arabidopsis, degradation of the Aux/IAA protein, IAA14, in response to auxin signaling, which activates the AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 along with a series of downstream transcription factors, also plays a critical role in this process. However, the specific mechanism by which auxin regulates callus formation remains unclear. By screening mutant library in the solitary root 1 (iaa14/slr) Arabidopsis background we obtained the callus formation related 2 (cfr2) mutant. The cfr2 mutant exhibited a stronger capacity for callus formation, as well as lateral root and adventitious root regeneration from leaf explants than wild type (WT) seedlings, but did not recover gravitropism capability. The auxin signal in cfr2 was significantly enhanced, and the expression of some downstream transcription factors was increased. Map-based cloning, whole genome resequencing, and phenotypic complementation experiments showed that the phenotypes observed in the cfr2 mutant were caused by a point mutation in the IAA14 promoter region. This mutation, which is predicted to disrupt the binding of LBD16, LBD19, and LBD30 to the IAA14 promoter, changed the expression pattern of IAA14 in cfr2. Taken together, our results identified a new mutation in the IAA14 promoter region, which affects the expression pattern of IAA14 and in turn its ability to control plant regeneration.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-024-01493-y.

Keywords: IAA14, Callus formation, Regeneration, Auxin, Promoter

Introduction

Plant cell totipotency is a well-known phenomenon wherein, under the appropriate conditions, plant tissues can regenerate new organs or even entire plants to cope with a changing environment (Reinert and Backs 1969; Sugimoto et al. 2011). Using this knowledge, an in vitro regeneration system was established on the basis of the plant hormone auxin-cytokinin paradigm, in which tissue explants are preincubated on auxin-rich callus-inducing medium (CIM) to produce pluripotent cells (referred to as callus) which lack characteristics of the origin tissue; this callus tissue is then are transferred to shoot-inducing medium (SIM) containing a high concentration of cytokinin, or root-inducing medium (RIM) containing a high concentration of auxin, to regenerate plant shoots or roots, respectively (Skoog and Miller 1957).

The mechanisms governing the auxin-induced callus formation that underpins this regeneration system are a prominent area of focus in plant regeneration research, and in recent years the molecular events controlling callus formation have been unveiled in great detail (Ikeuchi et al.2013). Using Arabidopsis root and aerial organ tissue explants, researchers have shown that auxin-induced callus formation originates from pericycle or pericycle-like cells via activation of the root development pathway, and the resulting callus tissue resembles that of root meristems, as determined by the expressions of root meristem marker genes (Atta et al. 2008; Che et al. 2007; He et al. 2012; Sugimoto et al. 2010). Additionally, destruction of pericycle tissue using the diphtheria toxin chain A (DTA) did not initiate lateral root and callus formation on CIM (Ikeuchi et al.2013). In Arabidopsis, there exists at least three pathways related to callus initiation in lateral roots: the IAA14-AUXIN RESPONSE FACTOR7/19 (IAA14–ARF7/19) pathway, the WUSCHEL-RELATED HOMEOBOX 11 (WOX11) pathway, and the CALMODULIN IQ-MOTIF CONTAINING PROTEIN (CaM–IQM)-mediated Ca2+ signaling pathway (Guo et al. 2018; Sheng et al. 2017; Zhang et al. 2022). Consistent with these findings, explants from the aberrant lateral root formation 4 (alf4) mutant exhibited inhibited pericycle cell division and suppressed callus formation on CIM (Shang et al. 2016; Smet et al. 2006; Sugimoto et al. 2010).

In the IAA14–ARF7/19 pathway, four downstream transcription factors of ARF7/19, LBD16, LBD17, LBD18, and LBD29, which interact with bZIP59, play key roles in auxin-induced callus formation by activating the FAD-BINDING BERBERINE (FAD-BD) (Fan et al. 2012; Xu et al. 2018). Moreover, ARF7/19 recruits ARABIDOPSIS TRITHORAX-RELATED 2 (ATXR2) and guides it to the promoters of the LBDs, and subsequently activates their expression by epigenetic modification (Lee et al. 2017). Under auxin treatment, ARF7/19-WRKY23 and LBDs-bHLH041 modules synergically establish callus pluripotency during the regeneration process by activating the expression of PLETHORA 1 (PLT1), PLT2, and WUSCHEL-RELATED HOMEOBOX 5 (WOX5) genes (Xu et al. 2023). Alternatively, recent studies showed that WUSCHEL-RELATED HOMEOBOX 11 (WOX11) activated directly by wounding-induced auxin accumulation acts upstream of LBD16 and LBD29 in the first step of the cell fate transition during de novo root organogenesis, and thus it is proposed that callus formation and de novo root organogenesis share the same genetic pathway at the initiation stage (Liu et al. 2014).

Most auxin-mediated plant growth and development processes, including callus and lateral root formation, are dependent on auxin/indole-3-acetic acid (AUX/IAA)-ARF signaling modules (Berleth and Sachs 2001; Smet et al.2010). Aux/IAA genes were reported to be up-regulated rapidly by auxin, and the Aux/IAA proteins, most of which contain four highly conserved domains (I, II, III, and IV), are short-lived nuclear proteins. Domain II is responsible for interactions with the SCFTIR1 ubiquitin-ligase complex and degradation of the Aux/IAAs (Abel et al. 1994; Kim et al. 1997). Gain-of-function mutations in domain II block these interactions and stabilize the Aux/IAAs, resulting in repression of ARF functions that affect auxin-induced growth (Ramos et al. 2001). The solitary root 1 (iaa14/slr) mutant, in which a gain-of-function mutation occurred in domain II of IAA14, inhibits the activity of ARF7/ARF19-LBDs and blocks auxin-induced early pericycle cell division required for lateral root and callus initiation, resulting in primary roots that lack lateral root or callus formation (Fukaki et al. 2002; Lee et al. 2009; Okushima and Tasaka 2007). Additionally, the slr-1 mutant also exhibits small leaves, short and thin inflorescence stems, no root hair formation, and a reduced gravitropic response (Fukaki et al. 2002).

To identify downstream regulators of auxin-induced callus formation, in particular regulators of the IAA14-ARF7/ARF19-LBDs-mediated pathway, we generated an EMS-mutagenized iaa14/slr population, which is described in our previous work (Shang et al. 2016). Here, a new mutant named callus formation related 2 (cfr2), that recovered lateral root and callus formation abilities was generated. Our data show that cfr2 mutant exhibits greater callus formation and de novo root organogenesis capabilities than the wild type (WT), resulting from a point mutation in the IAA14 promoter, which likely functions by altering IAA14 expression.

Materials and methods

Plant materials and cultivation

Arabidopsis ecotypes Columbia (Col) and Landsberg (Ler) were used in this study. A cfr2 mutant was isolated from EMS-mutagenized-slr populations. From 5500 M1 generation individuals and approximately 250,000 M2 generation individuals, we tried to obtain mutants with restored callus formation ability. The iaa14/slr, DR5pro:GUS, J0121, and ProWOX5::GFP-ER marker lines have been described in previous studies (Cui et al. 2013; Shang et al. 2016). The iaa14-1 mutant was obtained from the Arabidopsis Biological Resource Center (ABRC). Seeds were germinated and cultured on 1/2 MS medium (1/2 MS salts, 1% sucrose, 0.6-1% agar) and grown vertically at 22 ± 1 °C under a 16:8 light/dark photoperiod. Callus induction was conducted on CIM (B5 salts, 0.6% phytogel, 0.05 µg/ml kinetin, 0.2 µg/ml 2,4-D) for 4–9 days. For adventitious root formation, the first two rosette leaves were cut off from the 12 days old plants which were growth on 1/2 MS and cultured on 1/2 MS for 15 days (Liu et al. 2014).

Phenotypic analysis

To measure the primary root length, the seedlings were photographed and analyzed using ImageJ software. To count the number of callus initiated, CIM-treated plant roots were observed and counted using an optical microscope (Olympus) (Fan et al. 2012). The ratio of the callus initiation number to root length was analyzed statistically. At least 20 plants for each genotype were examined, and data are shown as mean ± SE (p < 0.05, Duncan’s Test, SPSS 21.0).

Cytological analyses

To observe lateral root initiation and root organogenesis, leaf explants of DR5pro::GUS lines in the WT, iaa14/slr, and cfr2 backgrounds were immersed in GUS assay solution for 2 h. To visualize the expression pattern of IAA14, seedlings were incubated in the GUS assay solution for 6 h. Subsequently, the GUS stained seedlings were rinsed according to a previously described method and analyzed using a DIC microscopy (Olympus) (Cao et al. 2024; Jefferson et al. 1988). Three independent transformant lines were selected for GUS staining observation, and one of them was presented in the article. Each histochemical analysis was repeated three times. GUS staining was quantified using ImageJ software following the previously described method (Ambastha et al. 2021; Manzano et al. 2013). And the optical density of GUS signal was present as integrated optical density (I.O.D).

To visualize the expression of the pericycle identity marker J0121 (Laplaze et al. 2005; Sugimoto et al. 2010) and the CIM-induced callus marker WOX5 (Shang et al. 2016; Sugimoto et al. 2010), seedlings were stained with 50 µg/ml of propidium iodide and observed under a Leica SP5 confocal microscope. The propidium iodide signal was detected by excitation with an argon laser at 488 nm and a spectral detector set at 600 nm for the emission. The GFP signals were excited by an argon laser at 488 nm, and the emission spectra were collected between 500 and 550 nm. For the subcellular localization of IAA14, the excitation and emission spectra for the GFP signal were set as described above (Cao et al. 2024). Three independent lines were selected for GFP observation, and one of them was shown in the article.

Gene expression analyses

Total RNA was extracted using the RNeasy plant mini kit (Qiagen). For cDNA synthesis, the Super-Script III first-strand cDNA synthesis system (Invitrogen) was used. RT-qPCR was performed using the SYBR Green PCR Master Mix on a Roche LightCycler 96; three biological replicates were used for each treatment. The ACTIN2 reference gene was used for normalization, using the 2 − ΔΔCT method to calculate relative expression. The primers used for the analysis are listed in Supplementary Table 2.

Material preparation for next-generation sequencing

From the F2 generation obtained by backcrossing cfr2 and iaa14/slr, 60 lines of lateral root and callus recovery mutants were precisely selected, and equal amounts of tissue were cut from each seedling and pooled. Tissues from iaa14/slr mutant tissues were collected similarly for use as the control sample. Genomic DNA was extracted from each sample, and the quality was assessed by agarose gel electrophoresis. Next generation sequencing was performed by the Genergy Bio-technology Company using the Illumina Hiseq 3000 sequencing platform by using a paired-end strategy.

Plasmid construction and Arabidopsis transformation

To demonstrate that the point mutation in the IAA14 promoter was responsible for the callus recovery of iaa14/slr, we constructed the pIAA14::IAA14, mpIAA14::IAA14, pIAA14::mIAA14, mpIAA14::mIAA14 constructs. pIAA14 and mpIAA14 represent the original and mutated promoters, respectively. This means these two promoters are amplified with the genomic DNA from the wild type and cfr2 mutants. IAA14 and mIAA14 represent the original and mutated IAA14 coding sequence, respectively. This means these two coding sequences are amplified from the cDNA from the wild type and iaa14/slr, respectively. Approximately 2000 bp of DNA sequence from the IAA14 or mIAA14 promoter and IAA14 or mIAA14 coding sequences were PCR amplified and cloned into pCAMBIA1300 using the Universal One Step Cloning Kit (YEASEN). Similarly, to analyze whether the point mutation altered IAA14 expression, approximately 2000 bp of DNA sequence from the IAA14 or mIAA14 promoter and IAA14 or mIAA14 protein coding regions were PCR amplified and cloned into the pMDC163 vector in frame with the GUS coding sequence, or into the pCAMBIA1300 vector in frame with the GFP coding sequence, respectively. All plasmids were introduced individually into Agrobacterium tumefaciens strain EHA105, then agro-transformed into Arabidopsis iaa14-1 by the floral dip method (Clough and Bent 1998). The primers used for plasmid constructions are listed in Supplementary Table 2.

Results

cfr2exhibits enhanced callus formation and de novo root organogenesis

To investigate the callus-forming and regeneration capabilities of the cfr2 mutant, 5 days old WT, iaa14/slr, and cfr2 seedlings were incubated on CIM for 4–7 days. Not only could the cfr2 mutant be recovered by root callus formation, it also exhibited enhanced callus formation relative to the WT (Fig. 1A and B). De novo root organogenesis is a type of regeneration in higher plants. Leaves cut between the petiole and blade from 12 days old cfr2 could regenerate many more adventitious roots than the WT, which could only form 2–3 adventitious roots per leaf explant when grown on hormone free medium for 15 days, meanwhile, only one adventitious root was formed by iaa14/slr leaf explants (Fig. 1C and D). These results showed that cfr2 recovered callus forming ability of the iaa14/slr mutant, and had stronger callus formation and de novo root organogenesis capabilities.

Fig. 1.

Fig. 1

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Enhanced callus- and adventitious root-forming capability in the cfr2 mutant. A Callus-forming phenotype of WT, iaa14/slr and cfr2. 5-day-old seedlings incubated on CIM for 7 days. B Quantitative analysis of the callus initiation on the primary roots of seedlings incubated on CIM for 4 days. C Adventitious root formation of WT, iaa14/slr and cfr2. Leaf explants cut from 12-day-old seedlings were cultured on 1/2 MS for 15 days. DAC represents days after culture. D Quantitative analysis of the adventitious root number per 15-DAC leaf explant cultured on 1/2 MS medium. Scale bars = 1 cm. At least six plants for each genotype were examined for Fig. 1B and D, and data are shown as mean ± SE (p < 0.05, Duncan’s Test, SPSS 21.0)

It has been shown that during de novo root organogenesis by leaf explants, mechanical injury leads to the accumulation of endogenous auxin in procambium cells at the wound site, and induces the expression of WOX11 and its homologous gene WOX12, thereby stimulating the cell fate transition (Liu et al. 2014). In this study, we used GUS staining to visualize the expression of DR5pro::GUS in leaf explants from WT, iaa14/slr, and cfr2 background lines during adventitious root formation (Fig. 2). Compared with the WT, the endogenous auxin accumulated to higher levels in the cfr2 mutant and the expression of WOX11 and WOX12 were induced significantly in cfr2 simultaneously. These results further indicate that cfr2 exhibits enhanced regeneration capabilities relative to the WT.

Fig. 2.

Fig. 2

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Increased auxin accumulation and induced WOX expression in the cfr2 mutant line during adventitious root formation. A GUS staining of 0-DAC, 2-DAC, 4-DAC, 8-DAC DR5pro:GUS leaf explants in WT, iaa14/slr and cfr2 background cultured on 1/2 MS. Scale bar = 10 μm (enlarged images of 0, 2, 4 and 8 DAC)/1 mm (leaf). B Relative expression level of WOX11 and WOX12 in Fig. 2A by real-time PCR. Error bars = ± SD, n = 3 biological replicates

Increased initiation of lateral roots in thecfr2mutant

Previous studies demonstrate that hormone-induced callus tissues resemble root apical meristems and form through ectopic activation of a lateral root initiation program (Sugimoto et al. 2010). In addition to the enhanced callus formation and adventitious root regeneration abilities, we also observed increased lateral root formation in the cfr2 mutant. As demonstrated by the kinetic curve analyses of lateral root growth, cfr2 developed many more lateral roots, including emerged and non-emerged primordia, than WT seedlings, however it did not recover the root hair formation or gravitropism phenotypes of the iaa14/slr line (Fig. 3A–F). Since it is well known that AUX/IAA-ARFs auxin signaling modules are correlated with lateral root founder cell specification and lateral root initiation, we analyzed the auxin response in DR5pro::GUS reporter lines. GUS staining showed that lateral root primordia were closely arranged to each other at a fixed distance in the cfr2 background, while only one primordium was detected in the WT background (Fig. 3G). Meanwhile, GUS signal accumulated in the root apical meristem and oscillation zones of the cfr2 mutant was 1.48- and 3.15-fold higher than that in WT, respectively (Figure S1 A, B). The enhanced DR5-GUS signal in the oscillation zone, which marked the regions for Lateral Root Primordium (LRP), could be responsible for the enhanced lateral root formation observed in cfr2 (Du and Scheres 2017).

Fig. 3.

Fig. 3

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Enhanced lateral root initiation in the cfr2 mutant. Morphology of 5-day-old WT, iaa14/slr and cfr2 seedlings. Insets show root hair formation of each seedling. B Phenotype of lateral root formation of 10-day-old WT, iaa14/slr and cfr2 seedlings. Scale bar = 1 cm. C, D Quantitative analysis of the primary root length C and lateral root primordia number per primary root (D) in 9-day-old WT, iaa14/slr and cfr2. E, F Kinematic analysis of primary root growth E and the average number of lateral root development (F) of WT, iaa14/slr and cfr2. Error bars = ± SD, n > 12. (p < 0.05, Duncan’s Test, SPSS 21.0). The plants were cultured vertically on 1/2 MS solid medium. G GUS staining of 7-day-old DR5pro:GUS seedlings in WT, iaa14/slr and cfr2 background. Scale bar = 10 μm. The solid arrowhead in G indicates lateral root primordium. Methods for GUS signal quantification are presented in the materials and methods section

RT-qPCR was used to quantify the expression of genes affecting lateral root formation, including GATA TRANSCRIPTION FACTOR 23(GATA23), LBD16, LBD17, LBD18, LBD29, LBD33, ALF4, and E2Fa in the WT, iaa14/slr and cfr2 backgrounds. The results showed that, compared with the WT, expression of GATA23, a key gene involved in cell specialization during lateral root initiation, and the LBDs, especially LBD33—an important functional gene involved in lateral root formation—were significantly up-regulated in cfr2 plants, supporting the observed increase in lateral root formation (Fig. 4).

Fig. 4.

Fig. 4

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Relative expression of genes involved in lateral root formation in the cfr2 mutant. WT, iaa14/slr and cfr2 seedlings were harvested at 12 days. Error bars = ± SD, n = 3 biological replicates

Cytological analysis of root and callus tissues

To determine the cytological basis of the recovery of lateral root and callus formation abilities by cfr2 mutant, differential interference microscopy (DIC) was used to observe differences between the root cells of WT, iaa14/slr, and cfr2 seedlings before and after CIM treatment. When cultured vertically on MS medium, lateral root primordia in cfr2 seedlings were closely arranged in a disordered pattern and the adjacent primordia were initiated in both the same and opposite directions (Fig. 5A), whereas lateral root primordia in the WT seedlings exhibited a regular left-right alternating pattern (Development). After CIM induction, the spaced callus was initiated from the root pericycle cells in WT seedlings, whereas the callus of cfr2 seedlings was more enlarged, indicating increased cell division occurred (Fig. 5A).

Fig. 5.

Fig. 5

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Cytological analysis of root and callus tissue in the cfr2 mutant. A Cytology of lateral root primordia and callus formation in 7-day-old WT, iaa14/slr and cfr2 seedlings. B, C Expression pattern of the pericycle cell identity marker J0121 B and the root stem cell marker WOX5 C in the maturation zone of 7-day-old WT, iaa14/slr and cfr2 seedlings on MS or incubated on CIM for 2 days. GFP signals from the mature zone of primary roots were overlaid with The GFP signal shown in green were overlaid with the signals stained with propidium iodide represented in red. Scale bar = 10 μm (DIC)/75 µm (Confocal). The solid arrowhead in A indicates lateral root primordia

Furthermore, we also quantified the expression of the pericycle cell identity marker J0121 in WT, iaa14/slr and cfr2 seedlings. Before CIM treatment, there was no difference in the expression of J0121, which was specifically expressed in the root pericycle cells of all seedling lines. After CIM treatment, the expression of J0121 was not altered in iaa14/slr seedlings, however the GFP signal became gradually weaker in the rapidly dividing initial cells of WT and cfr2 seedlings (Fig. 5B), suggesting that the callus tissue of cfr2 also originated from pericycle cells. We also examined the expression patterns of the CIM-induced callus marker WOX5, which specifically expressed in root Quiescent center (QC) (Shang et al. 2016; Sugimoto et al. 2010; Sarkar et al. 2007), in WT, iaa14/slr and cfr2 seedlings. As shown in Fig. 5C, after CIM induction, GFP signal was detected in the developing callus tissue, which was consistent with observations in WT callus, indicating the callus formed by cfr2 also had characteristics of the root apical meristem.

A point mutation in theIAA14promoter recovered the callus forming ability ofiaa14/slrin thecfr2mutant

Genetic analysis revealed that, when the cfr2 mutant was backcrossed with iaa14/slr mutant, the resulting F1 population exhibited the iaa14/slr mutant phenotype and were defective for callus formation, while the F2 population conformed to the separation rule of single recessive mutation which displayed a segregation of cfr2:iaa14/slr as 3:1 (Fig. 6A, B). Next, the cfr2 and iaa14/slr mutants were crossed with Col-0 or Ler, respectively. The F1 populations obtained from crossing the iaa14/slr line with the Col-0 or Ler lines exhibited defective callus formation. The F1 populations obtained from crossing the cfr2 and Col-0 or Ler lines exhibited the cfr2 phenotype, and members of the F2 population recovered the ability to form callus, with a segregation proportion of cfr2:WT as 3:1. These results showed that the mutation in cfr2 was the recessive trait in the background of homozygous iaa14/slr mutation background and the dominant trait in the background of heterozygous iaa14/slr mutation.

Fig. 6.

Fig. 6

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A single point mutation in the IAA14 promoter is responsible for callus formation recovery in the iaa14/slr line. A-B Genetic analysis of cfr2 mutant. LR represents lateral root, NLR represents no lateral root. The 7-day-old seedlings of Col, iaa14/slr, cfr2, cfr2 × slr F1, cfr2 × Col F1were cultured on CIM for 12 days. C Schematic illustration of the map-based cloning of CFR2. The mutation site of cfr2 is located in BAC FCA1. Markers are indicated in capital letters. Exons are shown as black boxes, grey boxes represent UTRs, small red triangles indicate the gain-of-function mutation in iaa14/slr mutant, big red triangle indicates the mutant site in cfr2 promoter. D Illustration of mutation distribution of cfr2 among Arabidopsis chromosomes. E Expression levels of IAA14 in WT and iaa14-1. FL represents full-length transcript; BL represents transcript before the T-DNA insertion site; C represents cycle. The T-DNA insertion site of iaa14-1 (CS25214) is indicated in small black triangle in A. F mpIAA14::mIAA14 iaa14-1 transgenic seedlings simulated the phenotype of cfr2. The phenotype of 7-day-old iaa14-1, iaa14/slr, cfr2, pIAA14::IAA14 iaa14-1, mpIAA14::IAA14 iaa14-1, pIAA14::mIAA14 iaa14-1 and mpIAA14::mIAA14 iaa14-1 transgenic seedlings cultured on CIM for 8 days. Scale bar = 10 mm. G Expression levels of IAA14 in WT, iaa14/slr and cfr2. Roots of WT, iaa14/slr and cfr2 seedlings were harvested at 12 days for qRT-PCR. Error bars = ± SD, n = 3 biological replicates

To localize the CFR2 gene, we mapped it to an approximately 5 Mb genomic region spanning two bacterial artificial chromosome (BAC) clones F25E4 (7.0 Mb) and F16G20 (12.2 Mb) on chromosome 4, using the F2 population from crossing the cfr2 crossed with the slr/Ler line (Fig. 6C). Since the gain-of-function mutation site of iaa14/slr was located at 8.35 Mb, and the two molecular markers were close to the mutation site, the fragments between the two markers were not replaced in the Ler ecotypes during background replacement. Therefore, in this study, the F2 population from crossing the cfr2 line with the Ler ecotype was used again for further fine localization. Plants with callus phenotypes similar to cfr2 were selected as candidate seedlings, which were identified as homozygous mutants belonging to the IAA14 background by SSLP markers located 7 Kb upstream of IAA14. Finally, we mapped the CFR2 locus to a ~ 300 Kb region between 8.10 Mb and 8.40 Mb long in the BAC clone FCA1, using molecular markers between 7.0 Mb and 12.2 Mb. After sequencing the genes in this region, we found that a C-to-T transition had occurred − 901 bp before the ATG codon in the upstream promoter region of IAA14.

Additionally, next generation sequencing technology was employed to clone the CFR2 gene. Sequencing results showed a signal peak on chromosome 4 (Fig. 6D), and no intragenic mutation in the region between 8.10 and 8.40 Mb. Therefore, it is suggested that the point mutation − 901 bp before the ATG in the IAA14 promoter may be the cause of callus recovery in the iaa14/slr line.

To provide further evidence for that recovery of callus formation in iaa14/slr is caused by this point mutation, we generated pIAA14::IAA14, mpIAA14::IAA14, pIAA14::mIAA14, mpIAA14::mIAA14 constructs and transformed these into iaa14-1 (Col-2 genetic background)—a loss-of-function T-DNA insertion mutant (CS25214) of IAA14 (Overvoorde et al. 2005), obtained from the Arabidopsis Biological Resource Center (ABRC)—and confirmed by RT-PCR (Fig. 6E). After incubating the transgenic plants on CIM, we found that transgenic plants harboring the mpIAA14::mIAA14 insert could fully reproduce the callus-forming phenotype of cfr2, whereas the transgenic plants with the pIAA14::mIAA14 insert were defective for callus formation, similar to the iaa14/slr line (Fig. 6F). These results further confirmed that the point mutation in the IAA14 promoter was the cause of lateral root and callus formation recovery in the iaa14/slr line.

To predict the effects of mutations in cfr2 on transcription factor motifs, we used PlantPAN 4.0 software to analyze the sequences 100 bp upstream and 100 bp downstream of the mutation site. The results (Supplementary Table 1) showed that the point mutations in the promoter region changed nine transcription factor binding sequences. In particular, three cis-elements—TFmatrixID_0051, TFmatrixID_1114, and TFmatrixID_1537—which are the binding sites of LBD16, LBD19 and LBD30, respectively, were lost in cfr2. Mutations in the promoter region usually affect gene expression levels. To determine the effects of the point mutation in IAA14 promoter on the IAA14 expression, RT-qPCR was performed for the WT, iaa14/slr and cfr2 lines. The results revealed that the expression levels of IAA14 in cfr2 was not significantly changed relative to that in the WT and iaa14/slr (Fig. 6G).

The point mutation in the IAA14 promoter altered IAA14 accumulation and localization

Considering that the point mutation did not change the transcription level of IAA14, we assessed the expression of the IAA14 protein. To test whether IAA14 protein accumulation and localization changed, pIAA14::IAA14-GUS/GFP, mpIAA14::IAA14-GUS/GFP, pIAA14::mIAA14-GUS/GFP, mpIAA14::mIAA14-GUS/GFP constructs were generated and transformed into Arabidopsis Col-0. After screening and identification of homozygotes, we found that the expression of mIAA14-GFP/GUS under the control of the IAA14 promoter resulted in defects in callus and lateral root formation, matching the iaa14/slr phenotype, whereas the expression of mIAA14-GFP/GUS under the control of the mIAA14 promoter contributed to the recovery of callus and lateral root formation, but resulted in defective root hair formation, matching the cfr2 phenotype (Fig. 7A–D). These results were consistent with our previous conclusions, further demonstrating that the mutation in the IAA14 promoter was responsible for the recovery of callus and lateral root formation in iaa14/slr.

Fig. 7.

Fig. 7

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Expression pattern and subcellular localization of IAA14. The upper part of insets show GUS observation images of 9-day-old pIAA14::IAA14-GUS/GFP (A), pIAA14::mIAA14-GUS/GFP (B), mpIAA14::mIAA14-GUS/GFP (C), mpIAA14::IAA14-GUS/GFP (D) transgenic seedlings after 6 h staining. The lower part of insets show GFP images of 9-day-old pIAA14::IAA14-GFP (A), pIAA14::mIAA14-GFP (B), mpIAA14::mIAA14-GFP (C), mpIAA14::IAA14-GFP (D) transgenic seedlings observed by laser scanning confocal microscopic. Scale bar = 1 mm (seedlings)/10 µm (enlarged images)/75 µm (GFP). Methods for GUS signal quantification have been mentioned in the materials and methods section

Accumulation of the IAA14/mIAA14 protein was measured using GUS and fluorescent signals. GUS staining showed that under the control of the IAA14 promoter, IAA14 protein was localized to the root stele and pericycle tissues in the elongation region, and also concentrated in lateral root initiation sites. Whereas under the control of the mIAA14 promoter, the GUS signal was relatively diffuse in lateral root initiation sites and concentrated in the pericycle. Conversely, the mIAA14 protein was strongly expressed in all root cell layers in the elongation zone and in the stele of matured tissues, when driven by the IAA14 promoter. However, when driven by the mIAA14 promoter, the mIAA14 protein accumulation was enhanced in all cell layers of the elongation zone, and was also distributed in the root stele, lateral root initiation site, and lateral root epidermal cells in the matured root tissues. According to the quantitative analysis of GUS signal, the protein accumulation was generally relatively high in the oscillating regions of pIAA14::IAA14-GUS, pIAA14::mIAA14-GUS and mpIAA14::mIAA14-GUS. However, there was relatively little protein accumulation in the oscillatory region of mpIAA14::IAA14-GUS (Figure S2A). In the apical region, pIAA14::IAA14-GUS and mpIAA14::IAA14-GUS signal intensities were low, whereas pIAA14::mIAA14-GUS and mpIAA14::mIAA14-GUS proteins accumulated relatively more (Figure S2B). Additionally, the GFP signal observed by laser confocal microscopy showed the signal was strongest in the nucleus, indicating the point mutations in the promoter of IAA14 did not affect its subcellular localization. Taken together, the point mutation in the promoter of IAA14 may change the accumulation and distribution of the mIAA14 protein, thus leading to the recovery of lateral root and callus formation.

Discussion

Auxin and auxin signaling components are intrinsically involved in controlling the formation of callus, lateral roots, and adventitious roots in plants (Hofmann 2014; Shin and Seo 2018). The pathways involved in CIM-induced callus formation and root development are highly conserved (Hofmann 2014; Sugimoto et al. 2010). The Arabidopsis IAA14 gene is thought to play an important role in these processes (Fukaki et al. 2002; Guo et al. 2018). Auxin induced degradation of IAA14 will release ARF7 and ARF19, after which ARF7 and ARF19 will interact with a large number of interacting proteins, thereby affecting the expression of downstream genes (Maitra Majee et al. 2020; Nguyen et al. 2023; Yoko Okushima and Tasaka 2007). In particular, LBD family proteins (LBD16, LBD17, LBD18, LBD29, LBD33, and LBD19), which are directly activated by ARFs, have a direct effect on callus formation. In the mutant cfr2, the lateral root and callus formation defects were recovered simultaneously, while the geotropism and root hair defects were not, indicating that the functions of different developmental processes controlled by IAA14 are not completely consistent. Additionally, the cfr2 mutant showed enhanced auxin signaling both in the roots of seedlings and during the initiation of adventitious roots from leaf explants.

The cfr2 mutation site was identified to localize to the IAA14 promoter region by whole genome sequencing. However, the result of the genetic analysis was very intertwined. In retrospect, the inconsistent results of genetic analyses using backcross (crossed cfr2 with iaa14/slr) and hybrid (crossed cfr2 with Col-0 or Ler) populations suggest that the mutation of the promoter in cfr2 altered the expression pattern of the IAA14 protein without negating the function of the correctly located IAA14 protein.

The expression patterns and protein stability of most AUX/IAA genes are regulated by auxin signaling (Luo et al. 2018). At present, most studies on AUX/IAA have focused on elucidating the interactions of AUX/IAA proteins with other factors or the binding of AUX/IAA proteins to DNA (Kim et al. 1997; Luo et al. 2023; Maitra Majee et al. 2020; Nguyen et al. 2023; Zhang et al. 2022). Recently, it has also been shown by promoter replacement experiments that the promoter activity of AUX/IAA genes is critical for their function (Muto et al. 2007). After all, mutations in promoter regions may affect the transcription levels, expression patterns, or subcellular localization of genes (de Vooght et al. 2009; Haberle and Lenhard 2016). It may also affect binding to upstream regulatory elements or affect the sliding of the translation initiation complex on the mRNA (Bozhilov et al. 2021; Cramer 2019). In this study, mutations in the IAA14 promoter region affected the expression pattern of IAA14. It was predicted that the binding sites for LBD16, LBD19 and LBD30 in the IAA14 promoter were disrupted by the point mutation. Coincidentally, LBD16 and LBD19 exhibit opposing characteristics in both expression pattern and function during callus formation (Fan et al. 2012; Liu et al. 2019). Although more detailed evidence is currently unavailable, we suspect that LBD16 and LBD19 may play a role in regulating IAA14 expression and function. Many studies have shown that LBD16 was involved in the formation of lateral and adventitious roots (Lee et al. 2019; Liu et al. 2018; Wu et al. 2024). The expression of LBD16 and WOX5 was activated by WUSCHEL-RELATED HOMEOBOX11 (WOX11) (Hu and Xu 2016; Sheng et al. 2017), while the expression of WOX5 was inhibited by LBD19 (Liu et al. 2019). Compared to the wild type, the expression WOX5 was not detected in iaa14/slr after CIM induction, but appeared to be stronger in cfr2. Considering that iaa14/slr and cfr2 mutants have completely opposite phenotypes with respect to lateral root and adventitious root formation in leaf explants compared to the WT, we speculate that LBD16 and LBD19 may have some involvement in the control of these two phenotypes.

Supplementary Information

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Supplementary material 1 (DOCX 213.5 kb)

Supplementary material 2 (DOCX 16.0 kb) (16.1KB, docx)
Supplementary material 3 (XLS 85.5 kb) (85.5KB, xls)

Author contributions

Huifen Cao and Xiao Zhang conceived the project and designed the experiments, performed the experiment and wrote the manuscript; Huifen Cao, Feng Li, Zhiping Han and Baopeng Ding conducted the experiments. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Natural Science Foundation of Shanxi Province (20,210,302,123,343), Ministry of Education supply and demand docking employment education project (20,230,100,563), Shanxi Province colleges and universities general teaching reform and innovation project (J20231011, J20231036), Shanxi Datong University teaching reform and innovation project (XJG2022210, XJG2022258), Shanxi Datong University Research Project (Yungang Study) (2021YGZX45), Shanxi Datong University Students Innovation and Entrepreneurship Project (XDC2022147, XDC2022157).

Data availability

The data sets generated and analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Ethics approval

Compliance with ethical standards.

Consent to participate

All authors participate to finish the work.

Consent for publication

All authors agreed for publication.

Footnotes

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Supplementary Materials

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Supplementary material 1 (DOCX 213.5 kb)

Supplementary material 2 (DOCX 16.0 kb) (16.1KB, docx)
Supplementary material 3 (XLS 85.5 kb) (85.5KB, xls)

Data Availability Statement

The data sets generated and analyzed during the current study are available from the corresponding author on reasonable request.

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