A Small Auxin-Up RNA Gene, IbSAUR36, Regulates Adventitious Root Development in Transgenic Sweet Potato

Small auxin-upregulated RNAs (SAURs), as the largest family of early auxin-responsive genes, play important roles in plant growth and development processes, such as auxin signaling and transport, hypocotyl development, and tolerance to environmental stresses. However, the functions of few SAUR genes are known in the root development of sweet potatoes. In this study, an IbSAUR36 gene was cloned and functionally analyzed. The IbSAUR36 protein was localized to the nucleus and plasma membrane. The transcriptional level of this gene was significantly higher in the pencil root and leaf.This gene was strongly induced by indole-3-acetic acid (IAA), but it was downregulated under methyl-jasmonate(MeJA) treatment. The promoter of IbSAUR36 contained the core cis-elements for phytohormone responsiveness. Promoter β-glucuronidase (GUS) analysis in Arabidopsis showed that IbSAUR36 is highly expressed in the young tissues of plants, such as young leaves, roots, and buds. IbSAUR36-overexpressing sweet potato roots were obtained by an efficient Agrobacterium rhizogenes-mediated root transgenic system. We demonstrated that overexpression of IbSAUR36 promoted the accumulation of IAA, upregulated the genes encoding IAA synthesis and its signaling pathways, and downregulated the genes encoding lignin synthesis and JA signaling pathways. Taken together, these results show that IbSAUR36 plays an important role in adventitious root (AR) development by regulating IAA signaling, lignin synthesis, and JA signaling pathways in transgenic sweet potatoes.

In general, the root expansion is not only regulated by endogenous phytohormones and genes, but is also affected by the external environment [9][10][11].Previous studies have demonstrated that auxins, mainly indole-3-acetic acid (IAA), play an essential role in the initiation of SR swelling, and auxin concentration increase with the increase inroot diameter in the early stage of root expansion [12,13].During the early stage of storage root development, the endogenous IAA content and SRD1 transcript level increased concomitantly, SRD1-overexpressing transgenic sweet potato plants cultured in vitro produced thicker and shorter fibrous roots than wild-type plants, suggesting an involvement of SRD1 during the early stage of the auxin-dependent development of the storage root [14].The early accumulation of IAA in the rooting zone stimulated the formation of ARs in cuttings [13,15].Many studies suggest that the genes related to phytohormone, IAA, jasmonic acid (JA), and lignin biosynthesis are widely used for clonal plant propagation in sweet potato SRs [15][16][17].
Plants can quickly sense and respond to changes in auxin levels, and these responses involve several major classes of auxin-responsive genes, including the auxin/indole-3-acetic acid (Aux/IAA) family, the auxin response factor (ARF) family, small auxin-upregulated RNAs (SAURs), and the auxin-responsive Gretchen Hagen3 (GH3) family [18].
SAURs are early auxin-responsive genes, with a large family in plants [19][20][21][22].Many studies suggest that the SAURs gene family plays important roles in root formation.The overexpressionof CsSAUR31 exhibited longer roots in cucumber [23].It revealed that OsSAUR11 positively enhanced the ratio of deep rooting in transgenic rice [24].The PagWOX11/12a-PagSAUR36 module significantly promoted AR development via the auxinsignaling pathway in transgenic poplar [25].However, it is unclear whether some SAURs participate in the development of ARs in sweet potato.
The initiation of SR bulking and the subsequent thickening process of sweet potatoes is a complex biological process, including the accumulation of morphogenesis and assimilation products.Such information does not reflect an accurate relationship between those ARs andstorage root development, and it is unclear how many of those ARs actually become SRs.Also, it is unclear whether SRs emanate from these initial ARs.Elucidating the molecular mechanisms of AR development can contribute to the root architecture.Here, we identified a candidate SAUR gene, IbSAUR36, which regulates AR development in sweet potatoes.The results suggest that IbSAUR36 may be a useful potential target for further molecular breeding of high-yielding sweet potatoes.

Plant Materials
Sweet potato cultivars Jishu25 (JS25), Jishu29 (JS29), and Xuzihsu 8(XZ8) were planted in the greenhouse of the Crops Research Institute, Shandong Academy of Agricultural Sciences, Jinan, China.JS25 and JS29 were used to isolate the IbSAUR36 gene and analyze the expression in different tissues, respectively.XZ8 has been used to characterize the gene functionas described by Yu et al. (2020) [26].The function of the IbSAUR36 promoter was identified using Arabidopsis thaliana (Columbia-0, wild type, WT).

Cloning and Sequence Analysis of IbSAUR36 and Its Promoter
The Trizol Up Kit (ET111, Transgen, Beijing, China) was used to extract the total RNA and then transcribe the first-strand cDNA with the PrimeScript TM RT reagent kit and gDNA Eraser kit (PR047A, Takara, Beijing, China).The full-length cDNA of IbSAUR36 was amplified from the first-strand DNA using the homologous cloning method with specific primers.The open-reading frame (ORF) of IbSAUR36 was analyzed with ORF Finder.The molecular weight and theoretical isoelectric point (pI) of IbSAUR36 were calculated with ProtParam tool (https://web.expasy.org/protparam/,accessed on 1 December 2023).Amino acid sequence alignment was analyzed using DNAMAN V6 software.The phylogenetic tree was constructed with MEGA 7.0 software with 1000 bootstrap replicates.Genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method.The genomic sequence and promoter sequence of IbASUR36 were amplified from genomic DNA using the homologous cloning method with specific primers.All the specific primers are shown in Supplementary Table S1.The cis-acting regulatory elements in its promoter were analyzed using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/,accessed on 1 December 2023).

Expression Analysis
The transcript levels of IbSAUR36 in leaf, stem, hair root, pencil root and storage root tissues of the 125-day-old field-grown plants of JS25 and JS29 was analyzed with quantitative real-time PCR (qRT-PCR).Furthermore, the 2-week-oldof JS25 and JS29 plants were stressed in Hoagland solutionwith H 2 O (control) and 100 mM IAA, and 100 mM MeJA, respectively, and sampled at 0, 1, 3, 6, 12 and 24 h after treatments for analyzing the expression of IbSAUR36.Ibactin (AY905538) was used to normalize the expression levels in sweet potato.All the specific primers are showed in Supplementary Table S1.

Function Analyse of IbSAUR36 Promoter
The promoter of IbSAUR36 was inserted into the DX2181 vector with the β-glucuronidase (GUS) gene, and then it was transferred into the Agrobacterium tumefaciens strain GV3101.The transgenic Arabidopsis plants were produced and further grown in pots to obtain T 3 seeds.The GUS activity in transgenic Arabidopsis was examined by GUS histochemical staining solution.After staining, tissues were cleared by replacing the staining solution with several changes of 70% (v/v) and 90% (v/v) ethanol as necessary.

Regeneration of the Transgenic Sweet Potato Plants
The coding region of IbSAUR36 was inserted into a pUBI.U4::IbSAUR36-CaMV35S::DsRed expression cassette pNRT expression vector.The constructs wereused for A. rhizogenesmediated transformation as described by Yu et al. (2020) [26].The transgenic roots were harvested with the help of red fluorescent protein (RFP).In order to explore the crosssectional structural characteristics of the root cells, the root tissues were collected from CK and transgenic lines for paraffin sections and dyed with sarranine and solid green.Theexpression level of IbSAUR36 was analyzed with qRT-PCR using primers in different roots (Supplementary Table S1).The phytohormone contents of the roots were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS).

RNA-Sequencing
To analyze the function of IbSAUR36, the roots of transgenic sweet potato and CK were used for RNA-sequencing (RNA-seq).The RNA-seq library was constructed using an Ultra RNA sample preparation kit (Illumina, San Diego, CA, USA).Fragments were sequenced using an Illumina HiSeq 2500 according to the standard method (Illumina).Total reads were mapped to the Ipomoeatrifida genome (sweet potato GARDEN (kazusa.or.jp)).Differentially expressed genes were identified using Cuffdiff with default criteria (fold change > 1.5) and an adjusted false discovery rate (p value < 0.05).Three independent biological replicates were used for the RNA-sequencing analysis.An analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was conducted according to database instructions (KEGG PATHWAY Database).The gene expression patterns were graphically represented in a heat map by cluster analysis using TBtools software v2.041.

Statistical Analysis
Three biological replicates were conducted and the data were presented as the mean ± SE.All were analyzed using Student's t-test (two-tailed analysis).Significance levels at p < 0.05 weredenoted bydifferent small letters, respectively.

Identification of IbSAUR36 and Its Promoter in Sweet Potato
We obtained a SAUR family gene from the previous comparative transcriptome analysis of root development in two sweet potato cultivars, JS25 and JS29.This gene was named IbSAUR36 by homology analysis.The 626-bp cDNA sequence of IbSAUR36 included 507-bp ORF and encoded a protein of 168 aa (molecular weight: 19.167 kDa) with a predicted pI of 10.98.Multiple sequence alignment showed that the new SAUR protein contained an auxininducible domain and has high homology with SAUR36 proteins from Ipomoea triloba (ItSAUR36, XP_031111161.1,96.4%), Ipomoea nil (InSAUR36, XP_019189087.1,93.5%), Capsicum annuum (CaSAUR36, XP_016557650.2,60.1%), Nicotiana attenuata (NaSAUR36, XP_019258606.1,57.6%), and Solanum tuberosum (StSAUR36, XP_006347888.1,56.4%) in the NCBI database (Figure 1A).Phylogenetic analysis of SAUR proteins with a neighbor-joining method revealed that the new SAUR protein has high homology with ItSAUR36 proteins from Ipomoea triloba (Figure 1B).The SAUR gene sequence is the same as that of IbSAUR36 (Ibat.Brg.04E_G002670.1)from the updated genome assembly and annotation for the sweet potato variety I. batatas Beauregard.Thus, the new SAUR gene was named IbSAUR36.The 1494-bp promoter region of IbSAUR36 contained several phytohormone-responsive, cis-acting regulatory elements (Supplementary Figure S1).

Expression Analysis
To study the potential function of IbSAUR36 in sweet potato, the expression was analyzed with qRT-PCR in different tissues and treatments of JS25 and JS29.For the field-grown plants, the expression level of IbSAUR36 was highest in the leaves and pencil roots (Figure 2A).The IbSAUR36 was downregulated in different developmental stages of the storage roots after 30 days (Figure 2B).The expression of IbSAUR36 peaked at 3 h after IAA treatment (Figure 2C) and downregulated after MeJA treatment (Figure 2D).These results suggest that IbSAUR36 might be involved in root development, IAA, and JA response pathways.
To study the potential function of IbSAUR36 in sweet potato, the expression was analyzed with qRT-PCR in different tissues and treatments of JS25 and JS29.For the field-grown plants, the expression level of IbSAUR36 was highest in the leaves and pencil roots (Figure 2A).The IbSAUR36 was downregulated in different developmental stages of the storage roots after 30 days (Figure 2B).The expression of IbSAUR36 peaked at 3 h after IAA treatment (Figure 2C) and downregulated after MeJA treatment (Figure 2D).These results suggest that IbSAUR36 might be involved in root development, IAA, and JA response pathways.

Functional Analysis of IbSAUR36 Promoter
The promoter region of IbSAUR36 contained several phytohormone-responsive cis-acting regulatory elements, including auxin, MeJA, SA, zein and ABA(Figure S1).The IbSAUR36 promoter fused to the GUS reporter gene has been introduced into Arabidopsis.GUS analysis of different tissues showed that IbSAUR36 was active in the young tissues of plants, such as the germinated seed, immature plant root, fruit, and bud of transgenic Arabidopsis (Figure 3).These results suggest that IbSAUR36 might be involved in tissue development through plant hormone pathways.

Functional Analysis of IbSAUR36 Promoter
The promoter region of IbSAUR36 contained several phytohormone-responsive cisacting regulatory elements, including auxin, MeJA, SA, zein and ABA (Figure S1).The IbSAUR36 promoter fused to the GUS reporter gene has been introduced into Arabidopsis.GUS analysis of different tissues showed that IbSAUR36 was active in the young tissues of plants, such as the germinated seed, immature plant root, fruit, and bud of transgenic Arabidopsis (Figure 3).These results suggest that IbSAUR36 might be involved in tissue development through plant hormone pathways.

IbSAUR36 Affected Phytohormone Homeostasis in Transgenic Sweet Potato Roots
To investigate the role of the IbSAUR36 gene in phytohormone signaling pathways of sweet potato, the overexpression vector pNRT-IbSAUR36 was constructed and introduced into sweet potato XZ8, while the XZ8 (CK) and the XZ8 introduced by pNRT (NRT) served as as negative controls.The RFP signal was observed in transgenic roots, but not in CK roots (Figure 4A).As shown in Figure 4B, the histological analysis of the transverse section revealed a higher number of lignified cells around xylem bundles in NRT roots than that of the CK lines.However, there was no obvious change in lignin deposition patterns in the roots between the IbSAUR36OE and CK lines.A qRT-PCR analysis revealed that the expression level of IbSAUR36 was significantly increased in IbSAUR36-overexpressingroots compared with that of CK and NRT roots (Figure 4C).Meanwhile, the IAA content significantly increased in IbSAUR36-overexpressing roots (Figure 4D).

IbSAUR36 Affected Phytohormone Homeostasis in Transgenic Sweet Potato Roots
To investigate the role of the IbSAUR36 gene in phytohormone signaling pathways of sweet potato, the overexpression vector pNRT-IbSAUR36 was constructed and introduced into sweet potato XZ8, while the XZ8 (CK) and the XZ8 introduced by pNRT (NRT) served as as negative controls.The RFP signal was observed in transgenic roots, but not in CK roots (Figure 4A).As shown in Figure 4B, the histological analysis of the NRT roots than that of the CK lines.However, there was no obvious change in lignin deposition patterns in the roots between the IbSAUR36OE and CK lines.A qRT-PCR analysis revealed that the expression level of IbSAUR36 was significantly increased in IbSAUR36-overexpressingroots compared with that of CK and NRT roots (Figure 4C).Meanwhile, the IAA content significantly increased in IbSAUR36-overexpressing roots (Figure 4D).

IbSAUR36 Regulates the Genes Involved in IAA, JA, and Lignin Signaling Pathways
To explore the mechanism of IbSAUR36 in the transgenic roots, differentially expressed genes and metabolic pathways in transgenic sweet potato were analyzed by RNA-Seq.Using CK and NRT as the control groups, we obtained a total of 8931 differentially expressed genes (DEGs), of which, 4344 genes were further analyzed by the KEGG database (Figure 5A).KEGG enrichment analysis showed that the 4344 DEGs in overexpressing roots were primarily enriched plant phytohormone signal transduction, phenylpropanoid biosynthesis, and starch and sucrose metabolism pathways (Figure 5B).

IbSAUR36 Regulates the Genes Involved in IAA, JA, and Lignin Signaling Pathways
To explore the mechanism of IbSAUR36 in the transgenic roots, differentially expressed genes and metabolic pathways in transgenic sweet potato were analyzed by RNA-Seq.Using CK and NRT as the control groups, we obtained a total of 8931 differentially expressed genes (DEGs), of which, 4344 genes were further analyzed by the KEGG database (Figure 5A).KEGG enrichment analysis showed that the 4344 DEGs in overexpressing roots were primarily enriched plant phytohormone signal transduction, phenylpropanoid biosynthesis, and starch and sucrose metabolism pathways (Figure 5B).

IbSAUR36 Regulated the Genes Involved in Root Development
The initiation and development of SRs are intricately regulated by a transcriptional regulatory network, including the biosynthesis of lignin, flavanols, and starch.To explore the mechanism of IbSAUR36 in the pre-swelling stage, we chose 9 storage-associated genes and analyzed their relative expression.The results showed that auxin pathway enzyme genes IbYUCCA6, IbTAR2, IbAUX1, and IbIAA26 were upregu- The DEGs of auxin biosynthesis (transcript14004, transcript18952) and import transduction (transcript7691) pathway were upregulated, while the auxin transferred gene (transcript14625), auxin outport gene (transcript23779), auxin signal transduction genes (transcript3105, transcript21118), JA-associated genes (transcript6686, transcript6860, tran-script4522 and transcript6161), and lignin biosynthesis genes (transcript10314, transcript22370) were downregulated (Figure 5C).The results indicate that overexpression of IbSAUR36 might regulate the IAA, JA, and lignin pathways.

IbSAUR36 Regulated the Genes Involved in Root Development
The initiation and development of SRs are intricately regulated by a transcriptional regulatory network, including the biosynthesis of lignin, flavanols, and starch.To explore the mechanism of IbSAUR36 in the pre-swelling stage, we chose 9 storage-associated genes and analyzed their relative expression.The results showed that auxin pathway enzyme genes IbYUCCA6, IbTAR2, IbAUX1, and IbIAA26 were upregulated, while the auxin glycosyltransferase gene IbUGT74, JA signal transduction enzyme gene IbJAZ, lignin biosynthesis enzyme genes Ib4CL and IbCAD, and root development-related transcription factor gene IbNAC83 were downregulated in IbSAUR36 overexpressing roots (Figure 6).lated, while the auxin glycosyltransferase gene IbUGT74, JA signal transduction enzyme gene IbJAZ, lignin biosynthesis enzyme genes Ib4CL and IbCAD, and root development-related transcription factor gene IbNAC83 were downregulated in IbSAUR36 overexpressing roots (Figure 6).

IbSAUR36 Is anImportant Factor in Auxin and JA Signaling Pathways
The phytohormone auxin and JA play essential regulatory roles in multiple aspects of plant growth and development and in stress responses [27][28][29].The YUCCA and TAR members are key enzymes of the IAA-biosynthetic pathway [30,31].Glycosylation of IAA is carried out by UDP-glycosyltransferase 84B1 (UGT84B1), while UGT74D1 is implicated in the glycosylation of the irreversibly formed IAA catabolite oxIAA in Arabidopsis [32].The content of IAA and expression levels of IbYUCCA6 and IbTAR2 were significantly increased, while those of IbUGT74 were significantly reducedin the IbSAUR36-overexpressing sweet potato roots (Figures 5, 6.Further, the auxin-responsive genes, including Aux/IAA, ARF, SAUR, and GH3, have roles in the tuberization process in sweet potatoes [33].Many studies have shown that the genes in the auxin signaling pathway are able to regulate the genes in the JA pathway.StARF16 regulates defense gene StNPR1 during the JA-mediated defense response upon necrotrophic pathogen interaction [34].The GH3 genes are cytosolic, acidic amido synthetases of the firefly luciferase group that conjugate auxins, jasmonates, and benzoate derivatives to a wide group The phytohormone auxin and JA play essential regulatory roles in multiple aspects of plant growth and development and in stress responses [27][28][29].The YUCCA and TAR members are key enzymes of the IAA-biosynthetic pathway [30,31].Glycosylation of IAA is carried out by UDP-glycosyltransferase 84B1 (UGT84B1), while UGT74D1 is implicated in the glycosylation of the irreversibly formed IAA catabolite oxIAA in Arabidopsis [32].The content of IAA and expression levels of IbYUCCA6 and IbTAR2 were significantly increased, while those of IbUGT74 were significantly reducedin the IbSAUR36-overexpressing sweet potato roots (Figures 5 and 6).Further, the auxin-responsive genes, including Aux/IAA, ARF, SAUR, and GH3, have roles in the tuberization process in sweet potatoes [33].Many studies have shown that the genes in the auxin signaling pathway are able to regulate the genes in the JA pathway.StARF16 regulates defense gene StNPR1 during the JA-mediated defense response upon necrotrophic pathogen interaction [34].The GH3 genes are cytosolic, acidic amido synthetases of the firefly luciferase group that conjugate auxins, jasmonates, and benzoate derivatives to a wide group of amino acids [35].SAURs and GH3 have been shown to be specifically induced by the plant hormone auxin and JA.The transcription factors MYC2 and SAUR21 play a central role in the hormonal balance between JA-Ile and IAA [36].The JA receptor JASMONATE-ZIM DOMAIN (JAZ) protein, JAZ4, has a prominent function in canonical JA signaling as well as the auxin signaling pathway [37].The SAUR family is an important factor in auxin signal transduction pathways, which include approximately 200 members in sweet potato [33].In this study, we characterized an auxin-regulated gene, IbSAUR36, in sweet potato.This IbSAUR36 gene was upregulated after IAA treatment and downregulated after JA treatment (Figure 2).The IbSAUR36overexpressing sweet potato root exhibited a better RFP signal and more accumulation of IAA (Figure 5).The genes in auxin and JA signaling pathways were upregulated in the IbSAUR36-overexpressing sweet potato roots (Figure 6).Based on all the above results, we propose that IbSAUR36 positively regulates the IAA and the JA signaling pathways.

IbSAUR36 Regulated the Lignin in Root Development
Numerous studies have confirmed that auxin plays a key role in AR formation [13,[38][39][40][41]. SAUR is the largest gene family with auxin-responsive factors [21].The main candidate genes, including MdSAUR2, MdSAUR29, MdSAUR60, MdSAUR62, MdSAUR69, Md-SAUR71, and MdSAUR84, regulated the root growth angle in apples [42].It was found that PbrSAUR13 promoted the synthesis and accumulation of stone cells and lignin, while Pbr-SAUR52 inhibited the synthesis and accumulation of stone cells and lignin [43].OsSAUR11 positively regulates rooting in rice through participating in the regulation of auxin signaling [24].The findings revealed high levels of CsSAUR31 expression within the root and male flower tissues, and CsSAUR31-overexpressing plants exhibited longer roots and hypocotyls in cucumbers [23].However, the function of the SAUR gene family in sweet potato is largely obscure.
Sweet potato is an important tuberous root crop, and its yield depends on a change in the developmental fate of ARs into SRs [2,44].The lignin biosynthesis limits sweet potato yield formation in storage roots [45].The AP2/ERF transcription factor, IbRAP2.4,inhibited SR formation in transgenic sweet potato by comprehensively upregulating lignin biosynthesis pathway genes [46].Transcriptional profiling of sweet potato roots indicates down-regulation of lignin biosynthesis and up-regulation of starch biosynthesis at an early stage of SR formation [47].The lignin biosynthesis genes IbPAL, IbC4H, Ib4CL, IbCCoAOMT, and IbCAD were upregulated under GA treatment in the sweet potato roots [5].MtCAD1 knockout medicago truncatula plants have reduced lignin content and display slower growth than WT [48].IbCAD1-overexpressing plants displayed lower root weights and lower ratios of tuberous roots to pencil roots than WT [49].In this study, overexpression of IbSAUR36 regulatedlignified cell development in ARs (Figure 4B).Further, the lignin biosynthesis genes Ib4CL and IbCAD were downregulated in the IbSAUR36-overexpressing sweet potato roots (Figure 6).The results show that IbSAUR36 may regulate the lignin biosynthesis in root pre-swelling development.

Conclusions
In this study, an IbSAUR36 gene was cloned and functionally analyzed.We demonstrated that the overexpression of IbSAUR36 promoted the accumulation of IAA, upregulated the genes encoding IAA synthesis and its signaling pathways, and downregulated the genes encoding lignin synthesis and JA signaling pathways.Taken together, these results show that overexpression of IbSAUR36 may impact root development by IAA signaling, lignin synthesis, and JA signaling pathways in transgenic sweet potato.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes15060760/s1, Figure S1: Promoter of IbSAUR36 showing different cis-acting regulatory elements associated with phytohormone responses.Table S1: Primers used in this study.

Figure 1 .
Figure 1.Multiple alignment (A) and phylogenetic analysis (B) of IbSAUR36with its homologs from other plants.

Figure 1 .
Figure 1.Multiple alignment (A) and phylogenetic analysis (B) of IbSAUR36with its homologs from other plants.

Figure 2 .
Figure 2. Relative expression of IbSAUR36.(A)Relative expression of IbSAUR36in different tissues of 125-day-old field-grown JS25 and JS29.L, leaf; ST, stem tip; YS, young stem; OS, old stem; PR, pencil root; SR, storage root.(B)Relativeexpression of IbSAUR36indifferent developmental stages of JS25 and JS29.d, day.(C,D)Relativeexpression of IbSAUR36in JS25 and JS29 after IAA or MeJA treatments.h, hour.The different small letters indicate a significant difference at p<0.05 according to Student's t-test.

Figure 2 .
Figure 2. Relative expression of IbSAUR36.(A) Relative expression of IbSAUR36 in different tissues of 125-day-old field-grown JS25 and JS29.L, leaf; ST, stem tip; YS, young stem; OS, old stem; PR, pencil root; SR, storage root.(B) Relative expression of IbSAUR36 in different developmental stages of JS25 and JS29.d, day.(C,D) Relative expression of IbSAUR36 in JS25 and JS29 after IAA or MeJA treatments.h, hour.The different small letters indicate a significant difference at p < 0.05 according to Student's t-test.

Figure 4 .
Figure 4. Production of the IbSAUR36-overexpressing sweet potato roots.(A)The root of transgenic sweet potato under red fluorescent protein(RFP) laser irradiation.(B) The histological analysis of transgenic sweet potato roots and CK.Bar =0.1 mm.(C)Expression analysis of IbSAUR36 in the transgenic roots and CK.(D) IAA content of transgenic sweet potato roots and CK.CK, roots of XZ8.NRT, roots of pNRT transgenic sweet potato.IbSAUR36OE, roots ofpNRT-IbSAUR36transgenic sweet potato.The data are presented as the means ± SEs (n = 3).The different small letters indicate a significant difference at p<0.05 according to Student's t-test.

Figure 4 .
Figure 4. Production of the IbSAUR36-overexpressing sweet potato roots.(A) The root of transgenic sweet potato under red fluorescent protein(RFP) laser irradiation.(B) The histological analysis of transgenic sweet potato roots and CK.Bar = 0.1 mm.(C) Expression analysis of IbSAUR36 in the transgenic roots and CK.(D) IAA content of transgenic sweet potato roots and CK.CK, roots of XZ8.NRT, roots of pNRT transgenic sweet potato.IbSAUR36OE, roots ofpNRT-IbSAUR36 transgenic sweet potato.The data are presented as the means ± SEs (n = 3).The different small letters indicate a significant difference at p < 0.05 according to Student's t-test.

Figure 6 .
Figure 6.Expression of the genes related to root development in IbSAUR36 overexpressing lines.CK, root of XZS8.NRT, root of pNRT transgenic sweet potato.IbSAUR36OE, root of pNRT-IbSAUR36 transgenic sweet potato.The data are presented as the means ± SEs (n = 3).The different small letters indicate a significant difference at p<0.05 according to Student's t-test.

Figure 6 .
Figure 6.Expression of the genes related to root development in IbSAUR36 overexpressing lines.CK, root of XZS8.NRT, root of pNRT transgenic sweet potato.IbSAUR36OE, root of pNRT-IbSAUR36 transgenic sweet potato.The data are presented as the means ± SEs (n = 3).The different small letters indicate a significant difference at p < 0.05 according to Student's t-test.4.Discussion 4.1.IbSAUR36 Is anImportant Factor in Auxin and JA Signaling Pathways