Functional Analysis of Aux/IAAs and SAURs on Shoot Growth of Lagerstroemia indica through Virus-Induced Gene Silencing (VIGS)

The plant hormone auxin plays an important role in cell division and the elongation of shoots to affect the plant architecture, which has a great impact on the plant yield, fruit quality and ornamental value; however, the regulatory mechanism of auxin controlling shoot growth is unclear in crape myrtle. In this study, two auxin/indole-3-acetic acid (Aux/IAA) genes and four small auxin upregulated RNA (SAUR) genes of auxin response gene families were isolated from dwarf and non-dwarf progenies of Lagerstroemia indica and then functionally characterized. Sequence alignment revealed that the six genes contain typical conserved domains. Different expression patterns of the six genes at three different tissue stages of two types of progenies showed that the regulation mechanism of these genes may be different. Functional verification of the six genes upon shoot growth of crape myrtle was performed via virus-induced gene silencing. When the LfiAUX22 gene was silenced, a short shoot phenotype was observed in non-dwarf progenies, accompanied by decreased auxin content. Therefore, we preliminarily speculated that LfiAUX22 plays an important role in the shoot growth of crape myrtle, which regulates the accumulation of indole-3-acetic acid (IAA) and the elongation of cells to eventually control shoot length.


Introduction
As a key component of plant architecture, plant height is one of the most important characteristics in crops, horticultural crops and ornamental plants, because it plays an important role in increasing the yield, improving the fruit quality, reducing management costs, and improving landscape effects [1][2][3]. At present, the studies on genes related to dwarfing traits have long been investigated in annual plants or model plants, such as rice [4,5], corn [6][7][8], wheat [6][7][8], while the mechanisms in woody plants remain poorly understood. For some dwarf plants, a short internode length is a key factor in the formation of dwarf woody plants, including dwarf peach [9,10], dwarf pear [11] and spur type apple [12]. Many researchers have pointed out that the shoot length is determined by the interaction of nitrogen before storage at −80 • C. One-year cuttings of dwarf and non-dwarf progenies were grown in a greenhouse at 22 • C for a 16 h light period with a relative humidity of 60%.

Cloning of LfiAUX/IAAs and LfiSAURs
For cloning the cDNAs of two LfiAux/IAAs and four LfiSAURs, the total RNA was isolated from shoot apices of dwarf and non-dwarf progenies using the RNAprep Pure Plant Kit (polysaccharides and polyphenolics-rich) (TIANGEN BIOTECH, Beijing, China). The cDNA was prepared from 1µg total RNA using the TIANScript RT Kit (TIANGEN BIOTECH, Beijing, China). The PCR reaction solution contained 1×EasyTaq PCR SuperMix (TransGen Biotech, Beijing, China), 0.2µM of each primer, and 100 ng cDNA. The cycling conditions were as follows: 94 • C; 2 min; 35 × 94 • C for 15 s, melting temperature (Tm) for 30 s, and 72 • C for 1 min; and 72 • C for 10 min. The primers are shown in Table S1. The PCR products were cloned into the pClone-EZ (CloneSmater, Houston, TX, USA) for sequencing.

Bioinformatics Analysis of Genes
The molecular weight (MW), number of amino acids, and isoelectric point (pI) for genes were estimated by using the online ProtParam tool (https://web.expasy.org/protparam/) [41]. Furthermore, amino acid sequence alignment of genes was done by using ClustalX2, and a phylogenetic tree was constructed by employing the neighbor-joining method from MEGA7 software.

Quantitative Real-time PCR
The total RNA was extracted from collected tissue, and the cDNA was then prepared from 1µg total RNA using the PrimeScript ® RT reagent Kit with gDNA Eraser (Perfect Real Time) (TAKARA, Shiga, Japan). The relative expression levels of genes were analyzed by PCR(CFX connect, Bio-Rad, Hercules, CA, USA). The qRT-PCR reaction solution (20 µL) contained 10 µL 2 × TB Green Premix Ex Taq II (TAKARA, Japan), 2 µL cDNA, 0.5 µL of each 10 mM primer, and 7 µL sterile distilled water. The primers are shown in Table S2. The elongation factor-1-alpha (EF-1α) (GenBank ID: MG704141) was used as the reference gene [42]. The 2 −∆∆Ct method was used to analyze the relative expression level of each gene.

VIGS Assay
pTRV1 and pTRV2 vectors were used as VIGS vectors [43]. The 445 bp cDNA fragment amplified through PCR using gene-specific primers with a EcoR I and BamH I site was used to generate a pTRV-LfiAUX22 vector. The primers are shown in Table S3. pTRV1, pTRV2, and pTRV-LfiAUX22 were transformed into Agrobacterium strain GV3101, respectively. After Agrobacterium strain GV3101 definitely containing three vectors was cultured in liquid lysogeny broth medium (LB) medium (100 µg/mL kanamycin, 50 µg/mL rifampicin, and 100 µg/mL gentamicin) for 12-16 h at 28 • C, the primary culture was resuspended into induction medium (100 µg/mL kanamycin, 50 µg/mL rifampicin, 100 µg/mL gentamicin, 10 mmol/L 2-Morpholinoethanesulfonic Acid (MES) at pH 5.6, and 20 µmol/L acetosyringone) for 12-16 h at 28 • C, followed by centrifugation for 2 min at 5000 rpm and supernatant was discarded. The pellet was resuspended in infiltration buffer (10 mmol/L MES at pH 5.6, 10 mmol/L MgCl2, and 200 µmol/L acetosyringone) to a final value of OD 600 = 2. The infiltration solution containing pTRV1 and pTRV-LfiAUX22 was mixed at a ratio of 1:1. The mixed infiltration solution was kept at the wound of young stems with the help of a 1 mL syringe. The plants infiltrated with the same ratio of infiltration solution containing pTRV1 and pTRV2 were used as a negative control. The infiltrated plants were cultured in the dark for 1 day and then returned to normal culture. The silencing of other genes was conducted using the same steps presented above. There were twenty one-year-old cuttings of non-dwarf progenies and thirty one-year-old cuttings of dwarf progenies used for VIGS. Twenty non-dwarf progenies were used as untreated plants

Endogenous IAA Content Measurement
The determination of endogenous hormone levels of IAA by the enzyme-linked immunosorbent assay (ELISA) technique was performed [44]. About 1 g of ground sample was homogenized in 2 mL 80% methanol (containing 1 µM butylated hydroxytoluene) and stored at 4 • C for 4 h. After centrifugation at 3000-4000 rpm for 10-15 min, the precipitate was extracted in 1.5 mL 80% methanol at 4 • C for 1 h. The two combined extracts transferred to C 18 Sep-Pak cartridges (Waters, Milford, USA) were dried under a stream of N 2 . Afterwards, the residue was dissolved in 0.01 m phosphate buffer solution (pH 7.5). The synthetic IAA ovalbumin conjugates in NaHCO 3 buffer (50 mM pH 9.6) were used to coat each well on the plates, and ovalbumin solution was used to wash the plates. The 50 µL samples and 5 µL antibodies were added to each well for a further 2 h. Then, 100 µL horseradish peroxidase-labelled goat antirabbit immunoglobulin was added to each well and incubated for 1 h. Finally, 100 µL o-phenylenediamine (OPD) substrate solution was added to each well and incubated for 10-30 min, after which the reaction process was stopped by adding 60 µL 3 M H 2 SO 4 to each well. The value of OD 400 was determined to calculate the hormone content. Calculations of the enzyme-immunoassay data were performed by an established standard curve [45].

Longitudinal Section Observation of Internodes
The stem segments' samples split along the median longitudinal from new shoots of untreated plants (WT), negative control plants (NC), and gene-silenced plants were fixed in FAA (70% ethanol: glacial acetic acid: 38% formaldehyde = 90:5:5) and then dehydrated, infiltrated, embedded, and stained. The treated samples were cut on a microtome (EM UC7, Leica, Wetzlar, Germany). The thickness of the paraffin sections was controlled at 6-12 µm and imaged with a microscope (Zeiss Axio Scope A1, Zeiss, Jena, Germany). The Nano Measurer software was used to calculate the cell length, and the average internode length was divided by the average cell length to obtain the average number of cells in the longitudinal section of the internode. Five biological replicates were performed for each sample. Data were performed via a one-way ANOVA.

Sequence of Two Aux/IAA and Four SAUR Genes in Lagerstroemia
According to our previous research, we screened out six genes differentially expressed in the auxin signal transduction pathway, of which two genes belong to the Aux/IAA family (LfiAUX22 and LfiIAA26), and four genes belong to the SAUR family (LfiSAUR26, LfiSAUR31, LfiSAUR39, and LfiSAUR50) [40]. There was no difference in the coding region sequences of these genes in the two types of progenies. LfiAUX22 and LfiIAA26 contained open reading frames of 597 and 792 bp which encoded proteins of 198 and 263 amino acids with a molecular mass of 21.9 and 28.7 kDa and pI of 5.47 and 5.09, respectively. The two genes were predicted to most likely be nuclear-localized in subcellular localization. Four SAUR genes contained open reading frames of 333-573 bp that encoded proteins of 110-190 amino acids, and the molecular weight ranged between 12.3 and 21.3 kDa, while the pI varied from 6.12 to 9.22. LfiSAUR26, LfiSAUR31, and LfiSAUR50 were predicted to most likely be nuclear-localized in subcellular localization, while LfiSAUR39 was predicted to be mitochondrial-localized (Table 1). The amino acid sequence alignment revealed that the LfiAUX22 protein contains four conserved domains (domain I-IV), which belong to the Aux/IAA family, while the domain IV of the LfiIAA26 protein is not complete (Figure 1). There were two types of nuclear localization signals (NLS) in LfiAUX22: one was a bipartite NLS located between the lysine-arginine (KR) motif and domain II, and another NLS was observed in domain IV. Additionally, there was only a binary NLS in LfiIAA26. The two NLS indicated that these two genes are located in the nucleus [46]. A phylogenetic tree constructed using Aux/IAA family genes from 13 plants and two Aux/IAA genes from Lagerstroemia showed that Aux/IAA family genes can be divided into 11 groups (A-K). LfiAUX22 was clustered together with PgrAUX22, EgrAUX22, AtIAA6, AtAUX22, AtIAA5, and VvAUX22 at group D, while LfiIAA26 was clustered together with PgrIAA26, SolIAA26, EgrIAA26A, EgrIAA26B, AtIAA26, and AtIAA18 at group F ( Figure 2). The amino acid sequence alignment of SAURs revealed that four LfiSAUR proteins contained a conserved domain (SAUR-specific domain, SSD), which belongs to the SAUR family and displays low homology in the N-and C-terminal regions ( Figure 3). A phylogenetic tree constructed using SAUR family genes from six plants and four SAUR genes from Lagerstroemia showed that SAUR family genes can be divided into six groups (A-F). LfiSAUR26 was clustered together with AtSAUR31, PtrSAUR32, and MdSAUR23 at group E, LfiSAUR50 was clustered together with AtSAUR39, AtSAUR70, MdSAUR4 and MdSAUR5 at group A; and LfiSAUR31 and LfiSAUR39 were clustered together with PgrSAUR50, MdSAUR15, and EgrIAA15A at group B ( Figure 4).

Expression of Six Genes in Different Tissues
The different expression characteristics of LfiAUX22, LfiIAA26, LfiSAUR26, LfiSAUR31, LfiSAUR39 and LfiSAUR50 were verified in shoot apex (SA), young leaf (YL) and shoot segment (SS) samples. LfiAUX22 and LfiIAA26 were highly expressed in the shoot segment; however, the expression level of LfiAUX22 in the three tissues of dwarf progenies was higher than in non-dwarf progenies, which is contrary to LfiIAA26. Different expression patterns of the four SAUR genes were observed in dwarf and non-dwarf progenies. LfiSAUR26 and LfiSAUR31 were highly expressed in the shoot segment, LfiSAUR39 was highly expressed in young leaves, and LfiSAUR50 was highly expressed in young leaves of dwarf progenies and the shoot segment of non-dwarf progenies. The expression level of LfiSAUR26 and LfiSAUR31 in the three tissues of non-dwarf progenies was higher than in dwarf progenies, which is contrary to LfiSAUR39, while the expression level of LfiSAUR50 in only two tissues of non-dwarf progenies was higher than in dwarf progenies, except in young leaves ( Figure 5).  Genes used in alignment are as follows:

Silencing the LfiAUX22 Gene Reduced the Shoot Length
At 2 months after infection, untreated plants and negative control plants definitively showed no significant changes in phenotype. After silencing LfiAUX22 and LfiSAUR39, only the new shoot length of LfiAUX22-silenced plants in non-dwarf progenies was significantly shortened; however, it was not as short as the internode length of dwarf progenies. The internode length of LfiAUX22-silenced plants was 85% that of non-dwarf progenies of untreated plants. After silencing LfiIAA26, LfiSAUR26, LfiSAUR31, and LfiSAUR50 in dwarf progenies, there were no obvious changes in the internode length of the treated plants (Figure 7). To further determine the changes in the cell pattern of the branches of the silenced plants, the cell number and cell length in the shoot segments of the candidate gene-silencing plants were counted by paraffin section technology. The results show that there were no changes in cell number and cell length in crape myrtle after silencing LfiSAUR39, LfiIAA26, LfiSAUR26, LfiSAUR31, and LfiSAUR50 genes; however, the silencing of LfiAUX22 shortened the cell length in the stem to 80.6% that of non-dwarf progenies of untreated plants (WT-S), although it did not change the cell number ( Figure 8).

Expression of Six Genes in Different Tissues
The different expression characteristics of LfiAUX22, LfiIAA26, LfiSAUR26, LfiSAUR31, LfiSAUR39 and LfiSAUR50 were verified in shoot apex (SA), young leaf (YL) and shoot segment (SS) samples. LfiAUX22 and LfiIAA26 were highly expressed in the shoot segment; however, the expression level of LfiAUX22 in the three tissues of dwarf progenies was higher than in non-dwarf progenies, which is contrary to LfiIAA26. Different expression patterns of the four SAUR genes were observed in dwarf and non-dwarf progenies. LfiSAUR26 and LfiSAUR31 were highly expressed in the shoot segment, LfiSAUR39 was highly expressed in young leaves, and LfiSAUR50 was highly expressed in young leaves of dwarf progenies and the shoot segment of non-dwarf progenies. The expression level of LfiSAUR26 and LfiSAUR31 in the three tissues of non-dwarf progenies was higher than in dwarf progenies, which is contrary to LfiSAUR39, while the expression level of LfiSAUR50 in only two tissues of non-dwarf progenies was higher than in dwarf progenies, except in young leaves ( Figure 5).

Silencing of Six Genes by VIGS
VIGS was used to understand the functions of two Aux/IAA genes and four SAUR genes. LfiAUX22 and LfiSAUR39 were silenced in non-dwarf progenies, and LfiIAA26, LfiSAUR26, LfiSAUR31 and LfiSAUR50 were silenced in dwarf progenies. At 40 days after infection, the virus was spread in plants infected with empty pTRV1 + pTRV2 and pTRV-candidate genes. qRT-PCR analysis was performed on the apical buds of untreated plants, negative control plants, and treated plants. The results show that the transcription abundances of the target genes in the treated plants were successfully reduced compared with untreated plants and the negative control plants by VIGS ( Figure 6).      Due to the important role of auxin in cell elongation, the endogenous auxin contents in the stem tips and stems of VIGS-silenced plants were determined. There were significant differences in the endogenous auxin content of the shoot apex and stem segments between non-dwarf progenies and dwarf progenies of untreated plants. Negative control plants definitively showed no significant changes in the endogenous auxin content. Among the six genes, only the silencing of LfiAUX22 caused a reduction in the endogenous auxin content in the stem segments, but not the shoot apex (Figure 9).
Forests 2020, 11, x FOR PEER REVIEW 13 of 18 caused a reduction in the endogenous auxin content in the stem segments, but not the shoot apex ( Figure 9).

Discussion
The Aux/IAA family is one of the most important auxin-responsive gene families in plants. Aux/IAA family members are short-lived nuclear proteins that contain four conserved domains encoded by the auxin early response gene family [50][51][52][53]. Domain I and Ⅱ play a key role in stabilizing the protein, and the "GWPPV" motif in domain Ⅱ is the core binding site of the TIR/AFB protein and auxin [54]. domain Ⅲ and Ⅳ are involved in the interaction of the Aux/IAA and auxin response factor (ARF) family proteins [55,56]. In this study, we successfully cloned LfiAUX22 and LfiIAA26. The two genes are predicted to be most likely located in the nucleus, and their protein sequence contains the four main conserved domains of the Aux/IAA protein (I-IV), which have been found to determine the interaction between Aux/IAA and the ARF protein [53,55]. LfiAUX22 contains four complete conserved domains, but the domain IV of LfiIAA26 is not complete. However, LfiIAA26 contains Dx (D/E) GD residues and conserved K residues in the Phox and Bem1p (PB1) domain, which affect the interaction between Aux/IAA and the ARF protein. The TIR1/AFB-Aux/IAA-ARF signaling system is a classic auxin signaling pathway, in which Aux/IAA proteins participate through interacting with ARF proteins as transcriptional repressors. At low auxin levels, Aux/IAA proteins are stable to directly dimerize ARF proteins, in order to inhibit the activation of downstream genes by ARF proteins. At a high auxin level, the interaction between Aux/IAA proteins and TIRA/AFB proteins promotes the degradation of the Aux/IAA protein leading to changes in target auxin response genes at the transcription level by released ARF proteins [57][58][59][60][61]. Therefore, the Aux/IAA proteins act as repressors in the auxin signaling system. Numerous experiments have established that the Aux/IAA genes are involved in the entire development process of plants, and some of them regulate cell expansion to affect the size and length of tissues. Many gain-of-function Aux/IAA mutants show shortened hypocotyls and curled leaves, and some loss-of-function Aux/IAA mutants show elongated hypocotyl, and lateral organ growth. These phenotypic changes are related to cell expansion [62][63][64][65][66][67]. According to previous studies and gene expression patterns in different tissues, it was found that LfiAUX22 and LfiIAA26 were mainly expressed in shoot segments, but their expression patterns were

Discussion
The Aux/IAA family is one of the most important auxin-responsive gene families in plants. Aux/IAA family members are short-lived nuclear proteins that contain four conserved domains encoded by the auxin early response gene family [50][51][52][53]. Domain I and II play a key role in stabilizing the protein, and the "GWPPV" motif in domain II is the core binding site of the TIR/AFB protein and auxin [54]. domain III and IV are involved in the interaction of the Aux/IAA and auxin response factor (ARF) family proteins [55,56]. In this study, we successfully cloned LfiAUX22 and LfiIAA26. The two genes are predicted to be most likely located in the nucleus, and their protein sequence contains the four main conserved domains of the Aux/IAA protein (I-IV), which have been found to determine the interaction between Aux/IAA and the ARF protein [53,55]. LfiAUX22 contains four complete conserved domains, but the domain IV of LfiIAA26 is not complete. However, LfiIAA26 contains Dx (D/E) GD residues and conserved K residues in the Phox and Bem1p (PB1) domain, which affect the interaction between Aux/IAA and the ARF protein. The TIR1/AFB-Aux/IAA-ARF signaling system is a classic auxin signaling pathway, in which Aux/IAA proteins participate through interacting with ARF proteins as transcriptional repressors. At low auxin levels, Aux/IAA proteins are stable to directly dimerize ARF proteins, in order to inhibit the activation of downstream genes by ARF proteins. At a high auxin level, the interaction between Aux/IAA proteins and TIRA/AFB proteins promotes the degradation of the Aux/IAA protein leading to changes in target auxin response genes at the transcription level by released ARF proteins [57][58][59][60][61]. Therefore, the Aux/IAA proteins act as repressors in the auxin signaling system. Numerous experiments have established that the Aux/IAA genes are involved in the entire development process of plants, and some of them regulate cell expansion to affect the size and length of tissues. Many gain-of-function Aux/IAA mutants show shortened hypocotyls and curled leaves, and some loss-of-function Aux/IAA mutants show elongated hypocotyl, and lateral organ growth. These phenotypic changes are related to cell expansion [62][63][64][65][66][67]. According to previous studies and gene expression patterns in different tissues, it was found that LfiAUX22 and LfiIAA26 were mainly expressed in shoot segments, but their expression patterns were the opposite in non-dwarf and dwarf progenies, indicating that these two genes might regulate stem development by different regulation mechanisms. Notably, the expression levels of LfiAUX22 in three tissues of non-dwarf progenies were higher than in dwarf progenies, but the auxin concentration in the shoot apex and internodes of no-dwarf progenies was higher than that of dwarf progenies [40]. In addition, the silencing of LfiAUX22 in the non-dwarf progenies only led to a reduction in the branch length, caused by a shortening of the cell length rather than a decrease in the number of cells. Furthermore, the silencing of LfiAUX22 only reduced the auxin content in the stem. Taken together, these results suggest that LfiAUX22 does not act as a repressor in the auxin signaling system. This is not common situation, but there are similar cases. In grapes, VvIAA19 affects cell division and cell elongation during fruit development. This gene may be a positive regulator of plant growth and development and is not induced by auxin [68]. One possible explanation is that this gene may play a positive regulation role in auxin synthesis. Therefore, this gene can regulate the auxin content to change binding of other Aux/IAA repressors with ARFs to regulate auxin-responsive development processes. In Arabidopsis, overexpression of the AtARF8 gene upregulates the expression of three GH3 genes in group II to adenylate the auxin IAA, which leads to the combination of IAA and amino acids causing a decrease in the auxin content and results in a short hypoblast axis [69]. Moreover, LfiAUX22 directly or indirectly represses the expression of negative regulators. Further investigations are needed to determine how LfiAUX22 regulates cell elongation. The silencing of LfiIAA26 in dwarf progenies did not cause changes in the phenotype and auxin concentrations. This might be because the silencing of a single gene usually fails to produce a phenotype due to extensive genetic redundancy among Aux/IAA family members [63,70,71], or because of the loss of function of this gene caused by the incomplete domain IV.
It is generally accepted that SAUR genes play an important role in cell elongation. In Arabidopsis, the overexpression of many SAUR genes causes cell elongation [28,29,31,32,72]. It has been found that SAUR proteins can interact with protein phosphatases of the PP2C.D family to regulate H+-ATPase activity and induce membrane acidification, and a low apoplastic pH then activates the cell wall expander protein to achieve cell growth. Furthermore, SAUR genes from different clades all exhibited this capacity [30,73,74]. Most SAUR genes that cause cell elongation are located in the plasma membrane, while some nuclear-localized SAUR genes inhibit cell elongation and do not respond to auxin, such as AtSAUR32 [35,75,76]. In this study, all SAUR genes except LfiSAUR50 were predicted to be located in the nucleus, and these four genes were all highly expressed in dwarf progenies, and lowly expressed in non-dwarf progenies. Among them, LfiSAUR26 is in the same clade as AtSAUR32, which inhibits cell elongation. In the VIGS experiment, these four genes did not cause phenotypic changes. This may because the silencing of a single gene did not cause the mutant phenotype due to the high degree of functional redundancy of the SAUR gene family. In addition, VIGS technology has a defect where it rarely completely inhibits the function of a gene. It is possible that target genes that are not completely silenced can produce enough functional protein and therefore cannot change the phenotype.

Conclusions
Auxin regulates transcription via the transduction pathway to coordinate plant growth and development and these responses involve several major classes of the Aux/IAA family, ARF family, SAUR family, and GH3 family. In this study, we successfully cloned two Aux/IAA genes and four SAUR genes and found that all genes except LfiIAA26 contain complete conserved domains. The VIGS of six genes in crape myrtle revealed that the silencing of LfiAUX22 reduced the auxin content and the elongation of internode cells in the shoot segments, which proved that LfiAUX22 is an important gene of the auxin signaling system for regulating the branch growth of crape myrtle. Although the silencing of other genes did not change the phenotype, their function requires further investigation.