ChIFNα regulates adventitious root development in Lotus japonicus via an auxin-mediated pathway

ABSTRACT

Adventitious roots (ARs), developing from non-root tissue, play an important role in some plants. Here, the molecular mechanism of AR differentiation in Lotus japonicus L. (L. japonicus) with the transformed chicken interferon alpha gene (ChIFNα) encoding cytokine was studied. ChIFNα transgenic plants (TP) were identified by GUS staining, PCR, RT-PCR, and ELISA. Up to 0.175 μg/kg rChIFNα was detected in TP2 lines. Expressing rChIFNα promotes AR development by producing longer roots than controls. We found that the effect was enhanced with the auxin precursor IBA treatment in TP. IAA contents, POD, and PPO activities associated with auxin regulation were higher than wild type (WT) in TP and exogenous ChIFNα treatment plants. Transcriptome analysis revealed 48 auxin-related differentially expressed genes (DEGs) (FDR < 0.05), which expression levels were verified by RT-qPCR analysis. GO enrichment analysis of DEGs also highlighted the auxin pathway. Further analysis found that ChIFNα significantly enhanced auxin synthesis and signaling mainly with up-regulated genes of ALDH, and GH3. Our study reveals that ChIFNα can promote plant AR development by mediating auxin regulation. The findings help explore the role of ChIFNα cytokines and expand animal gene sources for the molecular breeding of growth regulation of forage plants.

KEYWORDS:

• Chicken interferon alpha gene
• forage plants
• Lotus japonicus L.
• adventitious roots
• auxin regulation

1. Introduction

Adventitious roots (ARs) emerge from non-root tissues (such as tender stems and branches, etc.). AR is a crucial organ in forage plants and also plays an essential role in the uptake and utilization of nutrients and fixation in the soil¹, determining the growth rate, soil retention capacity, and so on. There are two ways to generate ARs. One is indirectly to form a new callus by mechanical damage to the wound indirectly; the other is to develop ARs directly from the organogenesis of self-cells (such as the formation layer, cortex, cylindrical sheath, or vascular bundle)². ARs, formed from stem bases near the cutting site subject to hydroponic induction, generally belong to the former type. This hydroponic rooting method is commonly used as a model for root research^3,4. The occurrence of ARs is a complicated organogenesis process involving the perception of multiple external environmental signals and is controlled by cascades coupling of internal and external cues⁵. Intracellular signaling molecules are mainly represented by hormones, including endogenous hormones such as auxin, cytokinin, gibberellin, abscisic acid, and ethylene^6,7. Among them, auxin is the most significant in regulating AR development¹. The synthesis, transport, and signaling of auxin play critical roles in AR development^2,8. ARs occur near the injury site of isolated organs, depending on the auxin accumulation in this region⁹. Differential auxin transport and accumulation at the stem bases lead to AR primordia formation in mutants of Solanum lycopersicum L⁸.

Four pathways were proposed for IAA biosynthesis using Tryptophan (Trp) in plants, including (i) tryptamine (TAM), (ii) the indole-3-pyruvic acid pathway (IPA), (iii) the indole-3-acetamide pathway (IAM), and (iv) the indole-3-acetaldoxime pathway (IAOX)¹⁰. More attention was mainly focused on the IPA and TAM auxin synthesis pathways in plants, which consist of many enzymes encoded by YUCCA, ALDH genes, etc. YUCCA can catalyze indole-3-pyruvate (IPA) to IAA in cells^11,12. ALDH participates in the oxidation of indole-3-acetaldehyde to IAA in IPA and TAM pathways¹³. Synthetic auxins are generally transported to the site to regulate AR development¹⁴. The auxin influx carrier AUX/LAX and the auxin efflux carrier PIN are involved in this process and play a significant role in auxin polar transport^15,16. The development regulation of auxin on roots mainly depends on the signaling transduction pathway. Genetic and biochemical studies have shown that AUX/IAA protein plays a core role in auxin signal transduction and is considered a suppressor of gene expression relevant to auxin inducing¹⁷. AUX/IAA is also a target gene for auxin-responsive factor (ARF) by the binding sites of its domains III and IV to ARF¹², and therefore causes a dual effect on auxin regulation^12,18. TIR is an auxin receptor; its SCF interacts directly with auxin to promote the degradation of transcriptional repressor AUX/IAA^19,20. A study shows this degradation is associated with identifying VGWPP sequences in AUX/IAA domains II to TIR1/AFB¹². ARF is a core gene in auxin signaling regulation, and is close to many downstream genes, such as GH3 and SAUR. ARFs regulate the early auxin-responsive genes with the help of AUX/IAAs²¹. When auxin concentration is low in cells, ARFs bind to AUX/IAAs repressor and inhibit the auxin-responsive genes transcription. However, when the auxin concentration is high, AUX/IAAs proteins are ubiquitinated by the binding of the TIR1-auxin-Aux/IAA complex. ARFs are released from the AUX/IAA-ARF dimer to activate downstream genes²¹. Early response genes (GH3 and SAUR) induced by ARFs will continue to transcribe and affect the auxin response under the high auxin concentration²². This regulatory activity of ARFs is carried out by its DBD domains, which can directly and specifically bind to auxin response elements (AuxREs) of GH3 and SAUR in auxin signal transduction^23–25.

Studies have shown that non-hormonal substances also regulate AR differentiation in plants. For example, high nitrate concentrations break the balance of endogenous hormones in apple brick wood, thereby inhibiting the AR formation²⁶. LLA (linoleic acid) and α-lna (α -linolenic acid) can stimulate the synthesis of biomass and secondary metabolites in ginseng, promoting root development²⁷. Organic germanium is a dietary supplement that enhances fresh and dry biomass accumulations of ARs²⁸. Aspergillus niger is involved in AR development by significantly enhancing the accumulation of salicylic acid (SA) and jasmonic acid (JA)^29,30. Piriformospora indica enhanced the root formation by inducing IAA biosynthesis in Dimocarpus longan Lour.³¹.

Interferon (IFN) is a multifunctional animal cytokine (CK), which has a variety of physiological functions such as hormone regulation, stress reduction, and immune regulation^32,33. Recombinant interferon proteins (rIFNs) in plants show physiological functions similar to those in animal cells^4,34. Studies on plants have shown that both human and animal IFNs and their inducers, 2’−5’oligosadenosine, have certain biological activities in plants^35,36. Plant root cytokinin activity was increased by introducing human interferon (rhIFN) in tobacco (Nicotiana tabacum L.) and wheat (Triticum aestivum L.)³⁷, indicating that IFN can promote root cell division and plant growth. It showed that the internode was shortened, the apical dominance was broken, and the root system had more branches after rhIFNα was introduced into lettuce (Lactuca sativa Linn.)³⁸. The early root system formed faster and developed better in transgenic tobacco with the introduced Chicken interferon alpha (ChIFNα) gene³⁹. Several studies have shown that animal IFN is involved in regulating plant development and differentiation, but its regulatory mechanism is still unclear.

AR differentiation of creeping forage plays an important role in pasture construction. L. japonicus is a high-quality forage and nectariferous plant. ChIFNα was introduced into L. japonicus in our previous study, and it was found that ARs differentiation and development were enhanced⁴. Here, we studied the AR phenotype, auxin-related enzymes, auxin content, and the expression of auxin-related genes based on the transgenic L. japonicus obtained in previous experiments⁴. It revealed the developmental effects and expressional regulatory mechanisms of ChIFNα in L. japonicus, which could guide the improvement of forage plants.

2. Materials and methods

2.1. Plant materials

Three transgenic lines (TP2, TP8, and TP10) were obtained from independent transformation events⁴. ChIFNα and GUS reporter genes were driven by CaMV 35S constitutive promoter in the transformed plasmid pSFRLH. The transgenic and wild-type parent plants (TP and WT) were planted in the Transgenic Plant Experiment Demonstration Base of Guizhou University (Guiyang, Guizhou, China, 106° 675′ E; 26° 427′ N).

2.2 DNA, RNA, and protein analyses materials

Genomic DNA from GUS-positive plants and WT was extracted using the plant DNA extraction kit (Tiangen, Beijing, China). The primers P1: 5’-GCTGTTCCAGCTTCTCCAC-3’ and P2: 5’-CCTGGTGTTTCCGGTAAGG-3’ were used to amplify 604 bp ChIFNα fragment following previous methods⁴. For RT-PCR identification of the ChIFNα gene, the total RNA was extracted by the plant RNA extraction kit (Omega, Norcross, Georgia, USA). cDNA strand was synthesized using the High-Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, Foster City, CA, USA). The ChIFNα fragment was amplified with the same primers. The total proteins were extracted from L. japonicus⁴⁰ and tested by the BCA protein concentration determination kit (Biyuntian, Shanghai, China). ChIFNα protein content in transgenic L. japonicus was detected according to the instructions of the R&B Chook IFN-α ELISA kit (Keming, Suzhou, China).

2.3 Observation of AR root development

To generate ARs, the branches of L. japonicus were isolated at the same growth stage from WT and TP with three to four internodes, and then fixed on a floating plate for hydroponics in a light incubator at 25°C. The culture light cycle was 16 h light and 8 h dark, and the light intensity was 15,000 LX. The cultured tap water (H₂O) was replaced daily to keep it fresh. To compare the difference in root length and number, the plants (WT and TP2, TP8, TP10) were treated, respectively, using H₂O and 0.4 mg/L IBA. Three replicates were made for different treatments, with 15 branches in each replicate. The differentiation of ARs from each growth stage was observed under the stereoscope (Analytik Jena, Gottingen, Germany). Root length and number were measured in centimeters on 15, 22, 29, and 36 d.

2.4. Measurement of IAA contents

To compare auxin content, exogenous ChIFNα (EC) treatment was also set up, where WT lines were cultured in water with the addition of 800 IU ChIFNα protein (Sango Biotech, Shanghai, China). 0.1 g apical buds and stem bases from water-cultured WT, TP2, and EC on 0, 3, 6, 9, and 12 d were sampled from three biological replicates and rinsed in ice-cold PBS (0.05 mol/L Tris-HCl, pH 7.4 phosphate buffer). According to Pan et al.,⁴¹ the extract was made from ground clean samples and diluted to 20% concentration. The IAA content of preparation was determined following the operating procedures of the indoleacetic acid (IAA) assay kit (Nanjing Jiancheng, Nanjing, China). Experiments were performed in at least three biological replicates.

2.5. Activity analysis of peroxidase (POD) and polyphenol oxidase (PPO)

WT, EC, and TP (TP2, TP8, and TP10) hydroponic leaves in the same period from 0 to 12 d were collected and ground for determining POD and PPO. The plant extract was prepared according to the method described by Zhao et al.⁴². And activities of POD and PPO were assayed using commercial kits (Suzhou Keming, Suzhou, China) in accordance with the manufacturer’s instructions. Experiments were performed in at least three biological replicates.

2.6. Differently expressed auxin-related genes from transcriptome analysis

Transcriptome data from apical buds and stem bases at 0 and 12 days during ARs differentiation from our previous study were analyzed⁴ (data deposited at https://ngdc.cncb.ac.cn/gsa/browse/CRA002426). Differentially expressed genes (DEGs) between the two varieties were determined using DESeq2 with a cutoff of a log2 foldchange (log2FC) |log2FC|≥1 and an adjusted p-value <0.05. Then, DEGs of each stage were further subjected to enrichment analysis of the Gene Ontology (GO) terms, using EggNog (v5.0.0.) database (http://eggnog5.embl.de/#/app/emapper). The results were visualized using bioinformatics tools (www.bioinformatics.com.cn). Auxin pathway genes of AR differentiation of L. japonicus in WT and TP2 were searched in expressed transcripts with the reference from KEGG functional databases (https://www.omicshare.com/to-ols/). TBtools software⁴³ was used to make heat maps of auxin-related DEGs.

2.7. Real-time quantitative PCR (RT-qPCR) detection

The auxin pathway of the transcriptome analysis was randomly selected for RT-qPCR verification. RNA was extracted, and cDNA was further prepared from apical buds and stem bases. Gene-specific primers were designed using Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA, USA) (Supplementary Table S1). RT-qPCR system and procedure were referenced in our previous research⁴. The Ljubiquitin gene was used as an internal reference to normalize the gene expression levels. The RT-qPCR analyses were performed using a qTower3G Real-time PCR System (Analytik Jena AG, Jena city, Germany) and SYBR® Green Fast Mixture (GenStar, Beijing, China). Normalized transcript abundances were calculated using the 2^−ΔΔCT method.

2.8 Statistical analysis

Microsoft Excel 2019 and Statistical Product Service Solutions (SPSS) Statistics 26.0 software (IBM Corporation, Chicago, Illinois, USA) were used for significance analysis.

3. Results and discussion

3.1 Identification of rChifnα transgenic plant

GUS staining was presented for shoots and roots of TP2, TP8, and TP10 (Figure 1a). PCR analysis of genomic DNA and RT-PCR indicated that ChIFNα was integrated into the genomic system and can be normally transcribed (Figures 1b & 1c). The ELISA protein analysis showed that expression levels of rChIFNα in TP2, TP8, and TP10 fresh leaves were 0.175, 0.170, and 0.165 μg/kg, respectively (Figure 1d). The TP2 had the highest rChIFNα content of 0.175 μg/kg, which was used for measurement of auxin content and transcriptome.

Figure 1. Identification of transgenic ChIfnα plants. (a) GUS staining of shoots and roots for wild type and transformed plant (WT: wild type; TP2, TP8, and TP10: different independent transgenic plants); (b) PCR analysis of TP in genomic DNA (M: DL2000 Marker; 1: SFRLH plasmid carrying ChIfnα gene; 2: H₂O; 3: Wild type (WT); 4–6: Different transformed lines containing ChIfnα gene); (c) PCR analysis of TP in genomic cDNA (RT-PCR); (d) ELISA detection of transgenes. Statistical analysis was conducted with software SPSS.     

3.2. ChIfnα promoting auxiliary roots growth associates with IBA

Auxiliary roots were generated from L. japonicus branches via hydroponic culturing. We examined the AR length and number from different treatments, including ChIFNα TP, IBA, and H₂O. An earlier AR development and faster AR elongation in TP than in WT were observed (Figure 2a). In the control with 0 mg/L IBA treatment, the length of roots in TP (TP2, TP8, and TP10) was significantly increased by an average of four times compared with that in WT (P < 0.05 or P < 0.01) (Figure 2b) on 15, 22, 29, and 36 d. However, ChIFNα expression does not affect de novo adventitious root numbers (Figure 2c). With 0.4 mg/L IBA treatment, the length of roots was significantly increased by an average of six times in TP2 (Figure 2d). More roots were observed on 15 d (P < 0.05) and 22 d (P < 0.05) with 0.4 mg/L IBA than those in WT (Figure 2e). Together, the results suggested that IBA and ChIFNα have affected much more on root length than on root number. This result suggested that increased root length of rChIFNα TP plants is associated with the IBA response.

Figure 2. Phenotypes of auxiliary roots (ARs). (a) The phenotype of ARs of wide type (WT) and transgenic lines (TP2, TP8, TP10) at 16 d in L. japonicus; (b) Average root length in H₂O treatment; (c) Average root count in H₂O treatment; (d) Average root length with 0.4 mg/L IBA treatment; (e) Average root count with 0.4 mg/L IBA treatment. Data were analyzed statistically using SPSS. *P < 0.05, **P < 0.01.

3.3. Auxin contents and enzymes activities in rChifnα transgenic plants

To understand the auxin levels in rChIFNα transgenic plants, IAA in apical buds and stem bases of AR early development were detected in TP2, WT, and EC-treated plants at a series of time points. ARs of TP2 and EC plants appeared on 12 d and 15 d, which was 3 d and 1 d earlier than WT, respectively (Figure 3a). IAA levels in apical buds of TP2 and EC plants were higher than WT, especially on 6 d (TP2, P < 0.05), 9 d (EC and TP2, P < 0.01), and 12 d (EC and TP2, P < 0.01) (Figure 3b), which were increased by 22.0%, 33.1%, 54.2% in TP2, and 6.7%, 13.9%, 24.1% in EC, respectively. IAA levels in stem bases of TP2 and EC plants were higher than WT, especially on 6 d (TP2, P < 0.05), 9 d (TP2, P < 0.05), and 12 d (TP2, P < 0.01) (Figure 3c), and increased by 11.4%, 23.3%, 31.8%, respectively. The higher IAA content indicated that ChIFNα may promote the synthesis and transport of auxin in the early stage of AR development. Since POD activity is associated with IAA regulation in the plant cell⁴⁴, POD activities in rChIFNα transgenic plants were detected with ELISA. The results showed that the POD activities were significantly higher in TP and EC plants than in WT from 3 to 12 d (Figure 3d, P < 0.05). The PPO activity is associated with auxin biosynthesis⁴⁵, so we also examined its activity. The PPO activities were higher in TP and EC plants than in WT from 3 to 12 d (Figure 3e), which reached a significant level on 3 d (EC and TP, P < 0.01), 6 d (EC and TP, P < 0.01), 9 d (EC, P < 0.05; TP, P < 0.01) and 12 d (EC, P < 0.05; TP, P < 0.01). These results suggested that ChIFNα affects the auxin regulatory network, including the enhanced PPO and POD activities.

Figure 3. IAA contents and enzyme activities in early adventitious roots (ARs). (a) Early auxiliary roots (ARs) observation of L. japonicus; (b) Auxin content of wide type (WT), exogenous ChIFNα (EC), and transgenic plant (TP2) in apical buds; (c) Auxin content of WT, EC, and TP2 in stem bases; (d) POD activity of WT, EC, and TP2, TP8, TP10; (e) PPO activity of WT, EC, and TP2, TP8, TP10. Data were analyzed statistically using SPSS. *P < 0.05, **P < 0.01.

3.4. GO Enrichment analysis of DEGs and auxin pathway genes screening

To explore the possible biological functions related to auxin, GO analyses were further performed to classify the DEGs (DEGs, more than two folds of change, P < 0.05) identified in the WT-0d vs TP-0d and WT-12d vs TP-12d comparisons (Supplementary Table S2). Figure 4 illustrates the main GO auxin terms of each comparison in the GO analysis. Seven auxin processes were identified in the comparisons of WT-0d vs TP-0d at apical buds, but no auxin process was found at stems at the same time. There were 14 auxin processes in the comparisons of WT-12d vs TP-12d at apical bud and five auxin processes in stem bases. These auxin processes included auxin transport, response to auxin, auxin-activated signaling, auxin binding and auxin biosynthetic process, and so on. These data indicated that ChIFNα’s promotion of adventitious root development may be related to auxin. Therefore, we will focus on the expression of auxin pathway genes. About 300 genes related to auxin pathways were annotated in the KEGG database of L. japonicus (Supplementary Table S3), including auxin synthesis gene family members ALDH (K00128), CYP83B1 (K11818), amiE (K01426), TAA1 (K16903) and YUCCA (K11816), auxin transport gene family members PIN (K13947) and AUX1 (K13946), and auxin signaling gene family members TIR1 (K14485), ARF (K14486), AUX/IAA (K14484), GH3 (K14487), and SAUR (K14488) (Figure 5a) (Supplementary Table S3). Among them, the expression levels of auxin pathway-associated DEGs were analyzed to reveal a difference of ARs in early development.

Figure 4. GO Enrichment analysis of DEGs in the auxin-related pathway. 0AB label corresponded to the 0 d in apical buds; 12AB label corresponded to the 12 d in apical buds; the 12SB label corresponded to the 12 d in stem bases.

Figure 5. Key gene families in auxin pathways and their transcript abundances in the RNA-Seq dataset. (a) Key gene families in auxin synthesis, transport, and signaling process and they are indicated by light red, blue, and green backgrounds respectively. (b) Expression profile of auxin synthesis DEGs in apical buds. (c) Expression profile of auxin transport DEGs in apical buds and stem bases. (d) Expression profile of auxin signaling DEGs in stem bases.

3.5. Regulation of DEGs responsible for auxin synthesis

Auxin biosynthesis in young tissues like shoots is transported to the stem bases to regulate the AR development^14,46. Here, auxin biosynthesis genes were focused on at the apical buds, while auxin signaling genes at stem bases, and transport genes in both. Compared with that in WT, in total, 7 and 11 auxin synthesis-related DEGs were identified in apical buds on 0 and 12 d, respectively (Supplementary Table S4). Two ALDHs were up-regulated on 0 d in TP compared with those in WT. In contrast, six DEGs (3ALDHs, 2CYP83B1s, and 1YUCCA) were up-regulated on 12 d in TP compared with those in WT (Figure 5b). Among them, the expression level of ALDH (Lj1g3v0838120) was 24 times higher in TP than in WT (marked by “★” in Figure 5b). The results showed that ChIFNα induced the expression patterns of auxin synthesis-related genes.

3.6. Regulation of DEGs responsible for auxin transport

Plant auxin in apical buds was downward transported to the stem bases where roots form⁴⁷. In total, 7 (2 PINs and 5 AUX1s) and 1 PIN (Lj0g3v0178879) auxin transport DEGs were identified from the transcriptome data at 12 d and 0 d in apical buds in TP compared with in WT, respectively, but only 1 PIN (Lj4g3v2139970) auxin transport DEG was found on 12 d in stem base, and none on 0 d (Supplementary table S5). At 0 d, the PIN (Lj0g3v0178879) was up-regulated in the apical buds of TP, while the other PIN (Lj4g3v2139970) was up-regulated in the stems of WT (Figure 5c). On 12 d of AR development, two PINs and five AUX1s were up-regulated in WT`s apical buds, but they were not found in TP. Therefore, an apparent change of auxin transport genes in apical buds and stem bases was observed in TP plants at the AR development.

3.7. Regulation of DEGs responsible for auxin signaling

DEGs involved in the auxin signaling pathway were also analyzed at stem bases. Four and 17 auxin signal-associated DEGs were identified at stems on 0 and 12d, respectively, from the transcriptome data compared with WT (Supplementary Table S6). Three DEGs were up-regulated genes in TP on 0 d, including one GH3 and two SAURs (Figure 5d). Six auxin signaling promoters (five GH3s, one SAUR) were up-regulated, and three transcriptional repressors (AUX/IAAs) were down-regulated in TP on 12 d. Among them, the expression levels of GH3 (Lj2g3v2027870) in TP were ~85 times that in WT (marked by “★” in Figure 5d). These results suggested that ChIFNα promotes AR development by regulating auxin signal-associated genes.

3.8. Validation DEG’s expressional regulation via quantitative real-time PCR

To validate the expressional regulation discovered in RNA-seq data, we randomly selected nine auxin pathways-related genes for RT-qPCR assays, including auxin synthesis genes Lj1g3v0838120 (LjALDH), Lj3g3v1475750 (LjCYP83B1) and Lj2g3v0674850 (amiE), signaling pathway genes Lj0g3v0144749 (LjSAUR), Lj5g3v1669330 (LjTIR1), Lj1g3v4407580 (LjGH3), Lj2g3v1468200 (LjAUX/IAA), and Lj4g3v0484820 (LjARF), transport genes Lj2g3v1589590 (PIN) (Figure 6). The PCR results showed that the regulations of these genes were consistent with those in the RNA-seq analysis.

Figure 6. RT-qPCR validation of auxin pathways genes for RNA-Seq. FPKM, fragments per kb per million fragments, is the abundance of genes determined from the transcriptome library sequencing data. Statistical analysis was conducted with software SPSS. *P < 0.05, **P < 0.01.

4. Discussion

4.1 ChIfnα interferes with auxin in ARs developmental regulation

Certain animal-originated genes can be expressed in plants and may affect plant growth. However, the mechanisms behind this are largely unknown. IFNα encodes cytokine with multiple functions in the animal. Its biological activities have been observed in transgenic tobacco, lettuce, and wheat plants^37–39. The effects of ChIFNα on tobacco^39,48,49 and grass⁴ were also observed in our previous studies. The ARs primordium of herbage formed early when treated with exogenous IFNα and overexpressing the ChIFNα gene. Whether this mechanism is associated with the auxin pathway has not been examined. Here, the ChIFNα impacts and molecular mechanism on ARs development were further explored with a focus on plant auxin. Exogenous IBA and target gene expressing significantly increased the ARs root length. This improved effect is similar to that from the auxin precursor IBA. We analyzed genes’ expression in plant auxin pathways and concluded that ChIFNα expression in the plant could promote ARs differentiation and development, interfering with plant hormone pathways. This interference may result from the cross-talk between cytokine and auxin pathways in plants⁵⁰.

IAA is one of the main ingredients of auxin⁵¹, and its biosynthesis occurs in rapidly dividing and growing tissues⁴⁶. Here, we found that IAA in apical buds was generally higher than in stem bases at the same period, indicating that the auxin synthesized in the apical buds was transported to the stem bases, which was consistent with previous research results^14,52. Our higher IAA content in plants of exogenous ChIFNα treatment (EC) and TP2 than in WT at 6, 9, 12 d (P < 0.01 or P < 0.05) suggested that ChIFNα enhanced the IAA synthesis. Meanwhile, more IAAs were accumulated in the stem bases where ARs occur. AR formation is regulated by a variety of hormones, among which auxin is the most crucial hormone^1,8. Therefore, ARs induction and formation are related to auxin content in Arabidopsis⁹ and tomato⁸. In our study, plant cells are more responsive to exogenous IBA and have a higher auxin content in apical buds and stem bases in EC and TP. Hence, the regulation effect of ChIFNα in ARs development is associated with auxin biosynthesis and transport.

EC and TP’s POD and PPO activities were higher than WT during ARs growing 6, 9, and 12 d (P < 0.01 or P < 0.05). POD can modify auxin content in plant cells and promote AR development⁴⁴. Studies have shown that PPO inhibits the production of polyphenols which hinder the auxin polar transport and promotes the development of ARs^53–55. These results suggest that ChIFNα may participate in the auxin pathway by increasing the activities of PPO and POD enzymes. Therefore, we speculate that the transport of POD and PPO regulates the root growth-promoting effect of ChIFNα.

4.2 ChIfnα regulates genes in the biosynthesis, signaling, and transport of auxin in transgenic plants

Many genes are involved in auxin pathways. This study identified many auxin-associated DEGs in TP plants compared with WT. Among those, ALDHs and YUCCAs for auxin synthesis were regulated at the apical buds in TP. The expression level of ALDH (Lj1g3v0838120) in TP was highly significant than in WT (>24 times). Previous studies have shown that ALDHs were involved in the TAM and IPA pathways for IAA synthesis¹³, and their expression was relevant to wheat IAA content⁵⁶. Overexpression of AtYUCCA1 in Arabidopsis promoted AR formation in the leaves by the IAA pathway⁵⁷. The formation of ARs callus was regulated via the TAM and IPA pathways of increasing OsYUC1 gene expression⁵⁸. Highly regulated ALDHs may play an important role in AR development. In addition, an up-regulated CYP83B1s was specially observed in TP. The cytochrome P450 enzyme could catalyze synthesis of IAOX from Tryptophan, which is the first step in IAA synthesis by the IAOX pathway⁵⁹. IAA homeostasis can be promoted in P450 CYP83B1 mutation and causes an excess of AR primordia in tomatoes⁶⁰. CYP83B1 catalyzes IAOX into indole glucosinolate biosynthesis via activating SUR1, contributing to IAA synthesis interruption^59,60. Maybe IAOX-IAA inhibitory pathway is further immobilized by ChIFNα for some reason in this study. We speculate that ChIFNα promotes auxin synthesis mainly dependent on the up-regulated ALDH and YUCCA in TAM and IPA pathways.

Polar auxin transport (PAT) affects root growth¹⁴ via AUX1s and PINs⁶¹. AUX1 can promote auxin transport in chrysanthemum⁵², affect AR numbers in Arabidopsis⁶², and promote primary root elongation in rice⁴³. PINs can promote auxin transport⁵² by making faster auxin move from the upper to the lower stem⁸. PIN2, PIN3, and PIN7 transport proteins have a significant contribution to the root growth of Arabidopsis⁶³. At least 27 PIN genes have been found in the L. japonicus genome⁶⁴. Still, only 21 have been annotated in the existing KEGG database. In our study, on 12 d of AR development, both PINs and AUX1s were up-regulated in WT`s apical buds but not in TP. Polar auxin transport proteins include AUX1s, PINs, ABCB, and PILs⁶¹. Auxin transport is not only determined by PAT but also by the transport in the phloem⁴⁷. Therefore, it is possible that the auxin is transported from apical buds to stems by ABCB and PILs proteins or the phloem. Hence, ChIFNα also affects auxin polar transport via regulating PINs and AUX1s.

It is known that auxin signaling pathway genes, including ARFs, AUX/IAAs, GH3s, and SAURs genes, are involved in AR development^65–67. The ARF is a kind of auxin-responsive gene⁶⁸. The study showed that ARF7 and ARF19 improved the regulation of lateral root primordia by up-regulating PUCHI expression in Arabidopsis thaliana⁶⁹. CaARFs were closely related to AR formation in Capsicum annuum⁷⁰, CsARFs to root development in Cannabis sativa⁷¹. AUX/IAA protein negatively regulates the ARF via forming ARF-AUX/IAA dimers^22,72 and mediating normal auxin signaling⁷³. GH3 is involved in root differentiation and development in rice^74,75 and Arabidopsis⁷⁶. The auxin response was affected by the transcription of AUX/IAA, GH3, and SAUR genes²². Overexpression of SAUR15 confers an increase in lateral roots⁷⁷. In our experiments, the regulation of these known genes was also observed. The expressed transgenic ChIFNα up-regulated multiple copies of GH3s and repressed the expression of multiple AUX/IAAs compared with that in WT. Especially, the significantly higher expression level of GH3 (Lj2g3v2027870) in TP (>85 times) suggests a strong regulation. Our results indicate that ChIFNα regulates the auxin response by up-regulating GH3 and reinforced AUX/IAA down-regulating in TP plants during AR development.

According to the auxin control program described by Figueiredo 2022⁷⁸ and other studies^{10–12,15,16,23–25}. We draw the regulatory figure of ChIFNα on plant AR development by mediating the auxin pathway (Figure 7). We find that ChIFNα promotes the synthesis of auxin in apical buds by up-regulating the expression of YUCCAs and ALDHs, key genes of TAM and IPA pathway, respectively, and these processes rely on the tryptophan synthesized in chloroplasts. However, it is interesting to note that CYP83B1, which inhibits auxin synthesis in plants, is also promoted by ChIFNα, which is worth exploring. Then, auxin is transported from apical buds to the stem bases, but PINs and AUX1 genes encoding auxin influx carrier AUX/LAX and auxin efflux carrier PIN, respectively, were inhibited in ChIFNα-transplanting plants, suggesting that ChIFNa altered auxin transport. In the auxin transduction pathway of ChIFNα-transplanting plants, on the one hand, ChIFNα promotes auxin content, binding to the receptor to inhibit the expression of transcription suppressor AUX/IAA; on the other hand, ChIFNα can directly inhibit the expression of AUX/IAA. Inhibition of AUX/IAA can promote the transcription of auxin early response genes (GH3s and SAURs), and the two genes have also been observed to be promoted in ChIFNα-transplanting plants. In conclusion, ChIFNα promotes AR development in L. japonicus by changing auxin synthesis, transport, and signaling pathways.

Figure 7. Diagram shows that ChIFNα regulates AR development through the auxin-responsive pathway. (“→” indicates promoting effect, “→” indicates indirect effect, and “→” indicates inhibiting effect).

5. Conclusions

Through transgenic identification, IAA detection, RNA-Seq, and other methods, the differentiation process of ARs was studied in L. japonicus transformed with ChIFNα. We found that ChIFNα expression promotes ARs development, IBA response, and auxin compound synthesis. Further study shows that ChIFNα effects expressional regulations involve enhanced auxin synthesis and signaling, transforming the transport process. The molecular regulatory mechanisms of ChIFNα in L. japonicus provide guides in the breeding improvement of forage grasses.

Author contributions

Conceptualization, P.W. and L.S.; methodology, V.L., and Q.G.; software, J.H., and Y.W.; validation, P.W., V.L., and Q.G.; investigation, P.W., and J.H.; writing – original draft preparation, P.W., X.W., and L.S.; writing – review and editing, X.W., L.S. and Y.W.; visualization, P.W.; X.W. and L.S.; funding acquisition, L.S. All authors have read and agreed to the published version of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15592324.2023.2218670

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

This research was funded by the National Natural Science Foundation of China grants (32260338) and (31660685) and the Guizhou Province Science and Technology Project (2023ZK119)