Genome-Wide Identification and Analysis on YUCCA Gene Family in Isatis indigotica Fort. and IiYUCCA6-1 Functional Exploration

Auxin is one of the most critical hormones in plants. YUCCA (Tryptophan aminotransferase of Arabidopsis (TAA)/YUCCA) enzymes catalyze the key rate-limiting step of the tryptophan-dependent auxin biosynthesis pathway, from IPA (Indole-3-pyruvateacid) to IAA (Indole-3-acetic acid). Here, 13 YUCCA family genes were identified from Isatis indigotica, which were divided into four categories, distributing randomly on chromosomes (2n = 14). The typical and conservative motifs, including the flavin adenine dinucleotide (FAD)-binding motif and flavin-containing monooxygenases (FMO)-identifying sequence, existed in the gene structures. IiYUCCA genes were expressed differently in different organs (roots, stems, leaves, buds, flowers, and siliques) and developmental periods (7, 21, 60, and 150 days after germination). Taking IiYUCCA6-1 as an example, the YUCCA genes functions were discussed. The results showed that IiYUCCA6-1 was sensitive to PEG (polyethylene glycol), cold, wounding, and NaCl treatments. The over-expressed tobacco plants exhibited high auxin performances, and some early auxin response genes (NbIAA8, NbIAA16, NbGH3.1, and NbGH3.6) were upregulated with increased IAA content. In the dark, the contents of total chlorophyll and hydrogen peroxide in the transgenic lines were significantly lower than in the control group, with NbSAG12 downregulated and some delayed leaf senescence characteristics, which delayed the senescence process to a certain extent. The findings provide comprehensive insight into the phylogenetic relationships, chromosomal distributions, and expression patterns and functions of the YUCCA gene family in I. indigotica.


Introduction
IAA (Indole-3-acetic acid) is the main type of natural auxin in plants, which plays important roles in a series of growth and developmental processes, such as embryonic polarization, vascular bundle differentiation, apical dominance, shoot architecture, root formation, phototropic response, gravity response, and senescence [1][2][3][4][5][6]. A growing body of evidence indicates that auxin participates in a variety of stress responses in plant, including high salt, low temperature, wounding, drought, and the like. Indeed, auxin can induce the rapid and transient high expression of auxin response genes ARF, Aux/IAA, GH3, SAUR, and LBD, which are involved in plant response to stress [7,8]. For example, with a firm foundation for further researches on their functions, adding to the data on YUCCA genes of Cruciferae.

IiYUCCA Gene Family Analysis in I. indigotica
13 YUCCA genes in total were identified in I. indigotica (Table 1) and the YUCCA genes were named from IiYUCCA1 to IiYUCCA11 (IiYUCCA1, IiYUCCA2, IiYUCCA3, IiYUCCA4, IiYUCCA5-1, IiYUCCA5-2, IiYUCCA6-1, IiYUCCA6-2, IiYUCCA7, IiYUCCA8, IiYUCCA9, IiYUCCA10, IiYUCCA11), referring to the homologous YUCCA sequences in A. thaliana. Among them, IiYUCCA6-2 was identified as a pseudogene, because there was a 2-bp deletion at the 450-bp position, which caused its translation to be terminated prematurely at the position of the 456-bp site when compared to IiYUCCA6-1. The CDS (Coding sequence) lengths of IiYUCCAs ranged from 1044 bp (IiYUCCA10) to 1335 bp (IiYUCCA6-1). Most IiYUCCA genes included three exons, while three genes (IiYUCCA2, IiYUCCA3, and IiYUCCA6-1) had four exons, and two genes (IiYUCCA1 and IiYUCCA4) had five exons. The theoretical isoelectric point (pI) and molecular weight (Mw) ranged from 8.36 to 9.58 (average pI = 9.03) and from 38.64 kDa to 49.77 kDa (average Mw = 46.28 kDa), respectively. The detailed information of each IiYUCCA gene is listed in Table 1, including the gene identifier (ID) in the genome database, the length of the amino-acid sequence, chromosomal location, and some basic physical and chemical properties.
Based on the genome data, the genetic mapping of IiYUCCA genes on the chromosomes (2n = 14) was investigated (Figure 1). Except for IiYUCCA6-1 and IiYUCCA6-2 (which were not mapped to any chromosomes), the other 11 IiYUCCA genes were randomly distributed on five chromosomes: Chr01, Chr02, Chr03, Chr06, and Chr07. The distributions of IiYUCCA genes on the different chromosomes were not uniform. Chr06 had four IiYUCCA genes, the highest number, while Chr02 and Chr07 both had only one. Furthermore, there were three IiYUCCA genes on Chr03. It should be noted that IiYUCCA5-1 and IiYUCCA5-2 (located on Chr01) and IiYUCCA3 and IiYUCCA9 (located on Chr06) were very close to each other, and whether they might have similar biologic functions or not still needs to be confirmed. Based on the genome data, the genetic mapping of IiYUCCA genes on the chromosomes (2n = 14) was investigated ( Figure 1). Except for IiYUCCA6-1 and IiYUCCA6-2 (which were not mapped to any chromosomes), the other 11 IiYUCCA genes were randomly distributed on five chromosomes: Chr01, Chr02, Chr03, Chr06, and Chr07. The distributions of IiYUCCA genes on the different chromosomes were not uniform. Chr06 had four IiYUCCA genes, the highest number, while Chr02 and Chr07 both had only one. Furthermore, there were three IiYUCCA genes on Chr03. It should be noted that IiYUCCA5-1 and IiYUCCA5-2 (located on Chr01) and IiYUCCA3 and IiYUCCA9 (located on Chr06) were very close to each other, and whether they might have similar biologic functions or not still needs to be confirmed.

Classification and Structural Analysis of IiYUCCA Genes
To clarify the structural diversities of IiYUCCA genes, an exon-intron composition map was drawn as shown in in Figure 2A. As expected, the gene family members in one branch exhibited similar intron-exon compositions, numbers, and lengths. For example, both IiYUCCA10 and IiYUCCA11 had three exons and similar coding sequence lengths, which indicated that they might have a close evolutionary relationship. Otherwise, 12 IiYUCCA genes (except for IiYUCCA6-2) were subjected to analysis with the MEME program to identify the conserved protein motifs. A total of 11 distinct motifs were found; among them, six motifs were identified. These were the FAD-binding motif, GC motif, ATG-containing motif 1, ATG-containing motif 2, FMO-identifying sequence, and

Classification and Structural Analysis of IiYUCCA Genes
To clarify the structural diversities of IiYUCCA genes, an exon-intron composition map was drawn as shown in in Figure 2A. As expected, the gene family members in one branch exhibited similar intron-exon compositions, numbers, and lengths. For example, both IiYUCCA10 and IiYUCCA11 had three exons and similar coding sequence lengths, which indicated that they might have a close evolutionary relationship. Otherwise, 12 IiYUCCA genes (except for IiYUCCA6-2) were subjected to analysis with the MEME program to identify the conserved protein motifs. A total of 11 distinct motifs were found; among them, six motifs were identified. These were the FAD-binding motif, GC motif, ATG-containing motif 1, ATG-containing motif 2, FMO-identifying sequence, and NADPH-binding motif. It was evident that the closely related proteins shared the same motif profiles, which suggested that the IiYUCCA proteins within one branch might share conservative functions. Moreover, it was not difficult to find that the conserved motifs were nearly the same, both in terms of number and type. However, there were some differences between IiYUCCA10 and IiYUCCA11. IiYUCCA10 lacked ATG-containing motif 2 and motif 8 ( Figure 2B). To some extent, the specific motifs might contribute to the functional divergences.

Phylogenetic Analysis of YUCCA Gene Family
In order to uncover the evolutionary relationships of YUCCA proteins among different species, we constructed the phylogenetic tree implemented in MEGA7.0, including A. thaliana, Brassica rapa, Brassica oleracea, Raphanus sativus, O. sativa, and I. indigotica (Figure 3). It was demonstrated that 96 YUCCA proteins from six species were grouped into four categories. Among them, Group I included five members, namely, YUCCA3, YUCCA5, YUCCA7, YUCCA8, and YUCCA9. The other groups contained two different members, such as YUCCA1 and YUCCA4 (Group II), YUCCA2 and YUCCA6 (Group III), and YUCCA10 and YUCCA 11 (Group IV). The phylogenetic analysis results apparently confirmed that YUCCAs of Cruciferae (dicotyledons) tended to be clustered into one branch, while O. sativa was separated from Cruciferae as monocotyledons. Therefore, it can be seen that the YUCCA proteins of I. indigotica were highly conserved evolutionarily. When the selection pressure was considered, the Ka/Ks ratio of the YUCCA gene family was 0.1126, far less than 1 (neutral selection) [33], which indicated a strong purifying selection on the YUCCA gene family for I. indigotica.

Expression Pattern Analysis on IiYUCCA Genes
The expression patterns of IiYUCCAs in different organs and different developmental periods (except IiYUCCA5-2 and IiYUCCA6-2) were carried out. Here, YUCCA5-2 and YUCCA6-2 were more likely to be regarded as pseudogenes; thus, we did not determine their expression levels, but this does not mean that they have no biological function. As shown in Figure 4, all IiYUCCA gene expressions varied significantly in nine organs. It was notable that IiYUCCA7 was specifically expressed in buds, which could play a key role in the bud development process. Interestingly, IiYUCCA6-1 was expressed at relatively high levels in most organs, indicating its importance in I. indigotica. IiYUCCA1, IiYUCCA9, and IiYUCCA11 were found to be predominantly expressed in the main roots, implying their specific functions in the root developmental process. IiYUCCA4 and IiYUCCA5-1 were mainly expressed in young leaves. IiYUCCA2 and IiYUCCA8 were expressed more highly in flowers than in other organs. Moreover, IiYUCCA3 and IiYUCCA10 were preferentially expressed in green siliques, reveling that they might participate in the reproductive development process. In summary, IiYUCCA members had various expression patterns in different

Expression Pattern Analysis on IiYUCCA Genes
The expression patterns of IiYUCCAs in different organs and different developmental periods (except IiYUCCA5-2 and IiYUCCA6-2) were carried out. Here, YUCCA5-2 and YUCCA6-2 were more likely to be regarded as pseudogenes; thus, we did not determine their expression levels, but this does not mean that they have no biological function. As shown in Figure 4, all IiYUCCA gene expressions varied significantly in nine organs. It was notable that IiYUCCA7 was specifically expressed in buds, which could play a key role in the bud development process. Interestingly, IiYUCCA6-1 was expressed at relatively high levels in most organs, indicating its importance in I. indigotica. IiYUCCA1, IiYUCCA9, and IiYUCCA11 were found to be predominantly expressed in the main roots, implying their specific functions in the root developmental process. IiYUCCA4 and IiYUCCA5-1 were mainly expressed in young leaves. IiYUCCA2 and IiYUCCA8 were expressed more highly in flowers than in other organs.
Moreover, IiYUCCA3 and IiYUCCA10 were preferentially expressed in green siliques, reveling that they might participate in the reproductive development process. In summary, IiYUCCA members had various expression patterns in different organs, and they could play different roles in the accumulation and regulations of auxin in different organs of I. indigotica.
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 1 Figure 4. The expression patterns of YUCCA genes in different organs from I. indigotica. 1-9: mature root, lateral root, mature stem, young stem, mature leaf, young leaf, fruit, bud, and flower, respectively. Different lowercase letters indicate the significant differences for the relative expression levels at p < 0.05 level.
In addition to differences for different organs, the expression patterns in differen developmental periods were also observed, and the four developmental periods (seven, 21, 60, and 150 days after germination) were detected. As shown in Figure 5, the expression levels of IiYUCCA2 IiYUCCA4, and IiYUCCA10 increased gradually, whereas the levels of IiYUCCA3, IiYUCCA5-1 IiYUCCA8, and IiYUCCA9 decreased with the developmental process. Moreover, IiYUCCA1 showed a trend of decreasing firstly and then rising. IiYUCCA1 expressions were detected only 2 days and 60 days after germination, whereas IiYUCCA6-1 was expressed highly compared to othe developmental periods. Therefore, these YUCCA genes had distinct spatiotemporal expression patterns in I. indigotica, laying a foundation for further function research on IiYUCCA genes. In addition to differences for different organs, the expression patterns in different developmental periods were also observed, and the four developmental periods (seven, 21, 60, and 150 days after germination) were detected. As shown in Figure 5, the expression levels of IiYUCCA2, IiYUCCA4, and IiYUCCA10 increased gradually, whereas the levels of IiYUCCA3, IiYUCCA5-1, IiYUCCA8, and IiYUCCA9 decreased with the developmental process. Moreover, IiYUCCA11 showed a trend of decreasing firstly and then rising. IiYUCCA1 expressions were detected only 21 days and 60 days after germination, whereas IiYUCCA6-1 was expressed highly compared to other developmental periods. Therefore, these YUCCA genes had distinct spatiotemporal expression patterns in I. indigotica, laying a foundation for further function research on IiYUCCA genes. Plants usually implement various signal regulatory networks to respond and adapt to differ tural environments. Thus, it was important to understand what kinds of factors controlled cific expressions of YUCCA genes and how they were regulated by different environmen nals. Due to the higher expression level in different organs, IiYUCCA6-1 was taken as an exam examine expression patterns in response to various stress treatments. As shown in Figure  UCCA6-1 showed a strong response toward PEG treatment, and the expression changes firs wed a sharp increase and then a rapid decrease. After 1 h of PEG treatment, the transcript leve UCCA6-1 was 1.13-fold higher than the control group. Additionally, the expression lev ulated by NaCl showed no obvious changes, with a slight increase (0.21-fold) after 6 h atment. Furthermore, IiYUCCA6-1 showed a completely opposite expression pattern wh osed to the wounding stimulation, and the expression level was 41% of the control group le er 1 h of treatment, before rising to 142%, demonstrating its sensibility to wounding stress. dition, IiYUCCA6-1 exhibited time-dependent and inconspicuous responses to Plants usually implement various signal regulatory networks to respond and adapt to different natural environments. Thus, it was important to understand what kinds of factors controlled the specific expressions of YUCCA genes and how they were regulated by different environmental signals. Due to the higher expression level in different organs, IiYUCCA6-1 was taken as an example to examine expression patterns in response to various stress treatments. As shown in Figure 6, IiYUCCA6-1 showed a strong response toward PEG treatment, and the expression changes firstly showed a sharp increase and then a rapid decrease. After 1 h of PEG treatment, the transcript level of IiYUCCA6-1 was 1.13-fold higher than the control group. Additionally, the expression levels stimulated by NaCl showed no obvious changes, with a slight increase (0.21-fold) after 6 h of treatment. Furthermore, IiYUCCA6-1 showed a completely opposite expression pattern when exposed to the wounding stimulation, and the expression level was 41% of the control group level after 1 h of treatment, before rising to 142%, demonstrating its sensibility to wounding stress. In addition, IiYUCCA6-1 exhibited time-dependent and inconspicuous responses to the low-temperature treatment, but only demonstrated a 0.58-fold change after 12 h of treatment. In summary, IiYUCCA6-1 exhibited the most obvious response to PEG treatment, but showed tiny variations when induced by low temperature. Therefore, the functions of the IiYUCCA6-1 gene were correlated with the kinds of stress resistance to some extent.

.5. Heterologous Expression of IiYUCCA6-1 in Nicotiana benthamiana
In this section, we further discuss YUCCA gene functions, taking IiYUCCA6-1 as an example iYUCCA6-1 was identified and cloned from I. indigotica (GenBank Accession Number: KY318462) ncoding a protein highly homologous to AtYUCCA6 (91%). The genome DNA of IiYUCCA6-1 was 835 bp, containing four exons and three introns. Its ORF (Open Reading Frame) length was 1335 bp ncoding 444 amino-acid residues. Detailed sequences and protein structure predictions are shown n the Supplementary Materials (Figures S1-S3). To determine the potential functions o iYUCCA6-1, the gene was transformed into N. benthamiana. After screening with Hygromycin B, 10 ransgenic lines (OE1-OE10) were tested at DNA and RNA levels. The detection results at the ranscriptional levels demonstrated that IiYUCCA6-1 was significantly expressed in 10 transgenic ines, and the expression levels of OE3 and OE9 were 200 times higher than in the control groups Figure 7A). Free IAA content was also investigated in all over-expressed lines, and the contents ncreased significantly, except for OE1 and OE4 ( Figure 7B), ranging from 5 to 6.5 μg/g. These results ere not completely consistent with the expression changes of IiYUCCA6-1, indicating tha iYUCCA6-1 may not be the only gene involved in the accumulation process of IAA. Moreover, as one-month-old control groups, OE3 and OE9 plants were selected to observe the henotypic differences. Obviously, the heights of OE3 and OE9 increased, and the whole plants ere thin and weak with prominent apical dominance ( Figure 7C), suggesting the typica yperauxin phenotype. Meanwhile, the expression levels of four early auxin response genes

Heterologous Expression of IiYUCCA6-1 in Nicotiana benthamiana
In this section, we further discuss YUCCA gene functions, taking IiYUCCA6-1 as an example. IiYUCCA6-1 was identified and cloned from I. indigotica (GenBank Accession Number: KY318462), encoding a protein highly homologous to AtYUCCA6 (91%). The genome DNA of IiYUCCA6-1 was 2835 bp, containing four exons and three introns. Its ORF (Open Reading Frame) length was 1335 bp, encoding 444 amino-acid residues. Detailed sequences and protein structure predictions are shown in the Supplementary Materials ( Figures S1-S3). To determine the potential functions of IiYUCCA6-1, the gene was transformed into N. benthamiana. After screening with Hygromycin B, 10 transgenic lines (OE1-OE10) were tested at DNA and RNA levels. The detection results at the transcriptional levels demonstrated that IiYUCCA6-1 was significantly expressed in 10 transgenic lines, and the expression levels of OE3 and OE9 were 200 times higher than in the control groups ( Figure 7A). Free IAA content was also investigated in all over-expressed lines, and the contents increased significantly, except for OE1 and OE4 ( Figure 7B), ranging from 5 to 6.5 µg/g. These results were not completely consistent with the expression changes of IiYUCCA6-1, indicating that IiYUCCA6-1 may not be the only gene involved in the accumulation process of IAA.

IiYUCCA6-1 Could Delay the Senescence Process
The fresh leaves of control and transgenic lines (OE3 and OE9) were isolated and treated w stress. The contents of chlorophyll and hydrogen peroxide and the expression of NbSA e detected. In Figure 8A, it is shown that the percentage of total chlorophyll content decrea ificantly in the transgenic lines and reduced the leaf yellowing process after seven day ness treatment, which suggests that the senescence process of transgenic plants was appare yed. Moreover, the content changes of hydrogen peroxide were further detected. As displa igure 8B, the increment in hydrogen peroxide content in transgenic lines was significantly lo in the control group, which might have also contributed to the delay in the senescence pro ansgenic plants. In addition, the expression level of NbSAG12, a senescence-related gene, ificantly lower than in the control group under darkness treatment ( Figure 8C). These find cate that the senescence process was obviously reduced when IiYUCCA6-1 was over-expres ansgenic tobacco. Moreover, as one-month-old control groups, OE3 and OE9 plants were selected to observe the phenotypic differences. Obviously, the heights of OE3 and OE9 increased, and the whole plants were thin and weak with prominent apical dominance ( Figure 7C), suggesting the typical hyperauxin phenotype. Meanwhile, the expression levels of four early auxin response genes, NbIAA8, NbIAA16, NbGH3.1, and NbGH3.6, were also observed, whose expressions were significantly higher than in the control groups ( Figure 7D). These results illustrated that the early auxin response genes were upregulated, responding to the increased IAA content, which was upregulated by the over-expressed IiYUCCA6-1 gene.

IiYUCCA6-1 Could Delay the Senescence Process
The fresh leaves of control and transgenic lines (OE3 and OE9) were isolated and treated with dark stress. The contents of chlorophyll and hydrogen peroxide and the expression of NbSAG12 were detected. In Figure 8A, it is shown that the percentage of total chlorophyll content decreased significantly in the transgenic lines and reduced the leaf yellowing process after seven days of darkness treatment, which suggests that the senescence process of transgenic plants was apparently delayed. Moreover, the content changes of hydrogen peroxide were further detected. As displayed in Figure 8B, the increment in hydrogen peroxide content in transgenic lines was significantly lower than in the control group, which might have also contributed to the delay in the senescence process of transgenic plants. In addition, the expression level of NbSAG12, a senescence-related gene, was significantly lower than in the control group under darkness treatment ( Figure 8C). These findings indicate that the senescence process was obviously reduced when IiYUCCA6-1 was over-expressed in transgenic tobacco.
antly lower than in the control group under darkness treatment ( Figure 8C). These fin that the senescence process was obviously reduced when IiYUCCA6-1 was over-exp genic tobacco.

YUCCA Numbers, Structures, and Phylogenetic Analysis in I. indigotica
As far as we know, most YUCCA genes in plants are gene families with multiple members, except for Petunia, which only has one YUCCA gene [35]. The YUCCA gene family was systematically described in A. thaliana, rice, maize, tomato, and so on [26,[36][37][38][39][40]. Our study established some descriptions for the comprehensive features of the YUCCA gene family in I. indigotica for the first time. There are 13 YUCCA genes in I. indigotica, which are distributed on five chromosomes, but IiYUCCA6-1 and IiYUCCA6-2 were not located. Although IiYUCCA6-1 and IiYUCCA6-2 are very similar, the latter is a pseudogene. Therefore, it was speculated that IiYUCCA6-1 might have vital functions because of the functional reduction of IiYUCCA6-2. Moreover, due to the similarities in both gene structures and conservative motifs, the IiYUCCA genes were deservedly clustered in one branch, showing the differences between I. indigotica and other plants (A. thaliana, B. rapa, B. oleracea, R. sativus, and O. sativa) based on the phylogenetic tree analysis. Furthermore, the IiYUCCA genes were divided into four subfamilies according to the conservative motifs. Meanwhile, the results showed that the YUCCA family members of several dicotyledons of Cruciferae were closely related and highly conserved in evolution, far from monocotyledon rice, consistent with the clustering analysis results of OsYUCCA gene family members in rice [41].

YUCCA Expression Patterns in I. indigotica
Gene expression patterns are usually consistent with their functions. Here, qRT-PCR analysis was conducted to investigate IiYUCCA gene expressions in nine organs and four developmental periods, which was conducive to assessing their possible biological functions. However, IiYUCCA5-2 was not quantified by qRT-PCR, because its prediction exon sequence showed a base deletion at the 960-bp position, suggesting an early translation termination, hinting that IiYUCCA5-2 might be a pseudogene. In addition, our transcriptome dataset of I. indigotica under different stress conditions (unpublished) showed no information for IiYUCCA5-2 ( Figure S4 shows its specific nucleotide sequence). Overall, there were distinct expression differences between the vegetative (IiYUCCA1, IiYUCCA4, IiYUCCA5-1) and the reproductive organs (IiYUCCA2, IiYUCCA7, IiYUCCA10). Furthermore, IiYUCCA9 and IiYUCCA11 showed similar expression patterns, implying that there could be a certain degree of functional redundancy, which also contributed to stabilizing the genetic networks [24]. It was reported that the expression profiles of YUCCA genes in P. trichocarpa [23], Lycopersicon esculentum [40], and Zea mays [27] displayed a wide scope of expression patterns in different kinds of tissues. However, the YUCCA genes of A. thaliana [13] and Fragaria vesca [42] were mainly expressed in the meristem, young primordium, vascular tissue, and reproductive organs. Thus, the different expression profiles reflected the species differences, and the differences in I. indigotica need to be discussed. Some IiYUCCA gene expressions were upregulated along with the developmental process, such as IiYUCCA2, IiYUCCA4, IiYUCCA10, and IiYUCCA11. In contrast, the expression levels of IiYUCCA2, IiYUCCA3, IiYUCCA5, IiYUCCA8, and IiYUCCA9 were downregulated. The results implied that these genes could play important roles in the developmental process. Therefore, the analyses of expression patterns in IiYUCCA genes provided some information to explore the functions of YUCCA genes in different organs and during different developmental processes, as well as contribute to speculating how spatio-temporal patterns of auxin biosynthesis are regulated in I. indigotica.
There was some evidence exhibiting that YUCCA genes performed potential regulatory functions under different kinds of stress conditions [17,43]. IiYUCCA6-1 was sensitive to NaCl, PEG, cold, and wounding treatments. Its changes in expression pattern reflected that this gene might be involved in complex regulatory processes, with a function in defense responses. Similar results were found in G. max, whereas C. sativus CsYUCCA8 and CsYUCCA9 responded significantly to high temperature, and CsYUCCA10b was sensitive to low temperature and salt stress [29]. GmYUCCA5 and GmYUCCA8 were proven to be sensitive to different stress stimuli, including drought, copper, zinc, low temperature, high temperature, and salinity [24]. These findings indicated the potential functions of YUCCA genes in improving the stress-resistance capacities in plants. It is necessary to confirm the expression patterns of YUCCA genes in I. indigotica, which contribute to enriching the YUCCA gene family characteristics and functional researches in plants.

Functional Analysis on IiYUCCA6-1 Ectopically Expressed in Tobacco
Auxin plays important roles in cell division, lateral root formation, flowering time, and leaf senescence in plants [43]. The functions of the YUCCA gene family in IAA biosynthesis, plant development, and leaf senescence are typically associated with IAA level changes [16]. In this study, the functions of IiYUCCA6-1 in the auxin biosynthesis process in I. indigotica were also discussed. Its functions in the IAA synthesis pathway were relatively consistent with those of AtYUCCA6 reported in A. thaliana [22] and strawberry [42]. It was found that over-expression of AtYUCCA6 increased not only IAA content, but also the expression levels of some auxin-responsive genes [22]. Here, similar results were obtained in transgenic tobacco, the expression levels of NbIAA8, NbIAA16, NbGH3.1, and NbGH3.6 were significantly higher than those in the control group. The results demonstrated that the over-expressed IiYUCCA6-1 gene might upregulate the auxin content, whereas the expression of early responsive genes was activated. Moreover, it was reported that YUCCA family genes could participate in plant resistance to stress [29], which was also proven in our experiment. Under dark stress, a visible change in total chlorophyll content in transgenic lines was observed, as well as H 2 O 2 content. Furthermore, the expression level of NbSAG12 decreased in the transgenic lines, which could be explained by the senescence process being delayed to a certain extent. Hence, the over-expression of IiYUCCA6-1 in tobacco could promote IAA content, as well as upregulate the expression of auxin biosynthesis genes, which caused the typical hyperauxin phenotype. Furthermore, with a decrease in the contents of total chlorophyll and H 2 O 2 , as well as a downregulation of NbSAG12, the leaf senescence process was delayed, and stress resistance could also be improved. However, there are more stress conditions that still need further research.

Isolation and Sequence Analysis of YUCCA Gene Family in I. indigotica
To identify YUCCA family genes in I. indigotica, the nucleotide sequences of YUCCA genes from A. thaliana, B. rapa, B. oleracea, R. sativus, and O. sativa were downloaded from TAIR (https://www. arabidopsis.org/), brassicaDB (http://brassicadb.org), and RGAP (http://rice.plantbiology.msu.edu/). The BLASTn program (aligned a given nucleic acid sequence to the database) with a threshold of 1 × 10 −20 was used to obtain the predicted sequences based on I. indigotica genome data. Then, Pfam (http://pfam.sanger.ac.uk/) and CDD (Conserved Domain Database) analyses were performed to confirm the encoded sequences of IiYUCCA proteins. GeneWise (Barcelona, Spain) was used to predict the pseudogenes. Furthermore, the physical and chemical properties (isoelectric point, molecular weight, GRAVY, and instability index) of IiYUCCA proteins were calculated using the ExPASy website (http://web.expasy.org/protparam/).
The chromosome locations of IiYUCCA members were investigated using MapChart software. The gene structures were analyzed using the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/, Peking University, Beijing, China). MEME software (http://meme-suite.org/, University of Nevada, Reno, USA) was used to find the common motifs, and the maximum number of motifs was set to 12 with the width of motifs set from six to 20. The multiple sequence alignments of YUCCA gene family proteins from six species were performed using the MUSCLE program (LynnonBiosoft Company, San Ramon, CA, USA) with default parameters. The full-length amino-acid sequences of 96 YUCCA proteins from six species were downloaded from their genome database, while the phylogenetic tree with aligned amino-acid sequences was constructed using MEGA 7.0 software with the maximum-likelihood method and verified with 1000 bootstraps. In addition, PAML 4.9d software was used to calculate the synonymous substitution rates (Ks) and non-synonymous substitution rates (Ka) of YUCCA genes in I. indigotica.

Plant Materials and Treatments
I. indigotica seeds were sown in pots (three plants each pot) in the greenhouse of the National Engineering Laboratory for Resources Development of Endangered Crude Drugs in Northwest China. The plants were cultivated at 23 ± 2 • C under a 16-h light/8-h dark photoperiod. Leaf materials at different developmental periods were taken from seedlings at seven days (cotyledon unfolding), 21 days (the second pair of euphylla unfolding), 60 days, and 150 days. Moreover, 60-day-old seedlings were treated with different abiotic stresses, namely, 5% PEG600, 100 mM NaCl, 4 • C, and 40% mechanical wounding, and the control group was irrigated with the same amount of deionized water. Then, all the leaf materials were collected after 0, 1, 3, 6, 12, and 24 h of treatment. The different organs, including roots, stems, leaves, buds, flowers, and siliques were taken from the plants in the field. All samples were quickly frozen in liquid nitrogen and stored at −80 • C for further research.

Gene Expression Analysis
Total RNA of each sample was extracted with TRIzol ® reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The quality of RNA was determined using a Nanodrop instrument (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis. The first-strand complementary DNA (cDNA) was synthesized using the PrimeScript TM II First Strand cDNA Synthesis Kit (TaKaRa, Dalian, China), with 1.0 µg of total RNA in 20 µL of water, stored at −20 • C. The RT-PCR products were diluted (1:20) for qRT-PCR analysis, which was performed using the SYBR ® Premix Ex Taq™ II (TaKaRa, Dalian, China) on a Roche LightCycler ® 96 System (Roche Diagnostics, Basel, Switzerland). All primer sequences for qRT-PCR are listed in the Supplementary Materials (Table S1). The endogenous reference genes from I. indigotica, IieIF2, IiPP2A-4, and IiRPL15, were used as the internal controls [44]. The relative gene expression levels were examined using the 2 −∆∆CT method. In each experiment, three biological and technical repeats were conducted.

Generation of Transgenic N. benthamiana
IiYUCCA6-1 was cloned by the specific primers (forward (F): 5 -ATGGATTTCTGCT GGAGGA-3 , reverse (R): 5 -TCAACCGTCTAGTAATCTACTAT-3 ), with the following PCR conditions: 94 • C, 3 min; 30 cycles of 94 • C, 30 s, 57 • C, 30 s, 72 • C, 2 min; 72 • C, 10 min. Subsequently, the product was cut with the restriction enzymes, Bgl II and Spe I, then inserted into the pCAMBIA1302 vector with the CaMV35S promoter. Furthermore, the vector was transformed into Agrobacterium tumefaciens strain GV3101, and the transformation of N. benthamiana was conducted using Agrobacterium-mediated leaf disc method. After screening by Hygromycin B, the over-expressed IiYUCCA6-1 plants were sampled separately. One part of the plants was used to extract the genomic DNA, and the other part was used to extract RNA and reverse-transcribe to cDNA. Then, the transgenic lines were detected at the DNA and RNA level.

Heterologous Transformation of IiYUCCA6-1 to N. benthamiana
The fresh leaves from control and transgenic lines were selected for the gene function analysis. The cDNA was used to detect the expressions of early responsive genes (NbIAA8, NbIAA16, NbGH3.1, NbGH3.6), and free IAA contents were measured with the help of a Plant Indole-3-acetic acid ELISA Kit (Enzyme-linked Biotechnology Co., Ltd., Shanghai, China), following the manufacturer's instructions. Next, some leaves were soaked in 3 mM MES (2-(N-morpholine) ethanesulfonic acid) medium (pH 5.7), and then placed in the dark. The total contents of chlorophyll and H 2 O 2 (Jiancheng, Nanjing, China), and the expression level of senescence related gene NbSAG12 (F: 5 -ATGAAGATGTGCCAGCG AAC-3 ; R: 5 -AGCTACTCCCGTCTATTGCC-3 ) were detected after dark treatments (zero and seven days), with NbActin (F: 5 -CGTTATGGTTGGAATGGGACAGAA-3 ; R: 5 -AAGAACAGGGTGCTCCTC GTGG-3 ) as the reference gene.

Statistical Analysis
Statistical analysis was performed with SPSS 22.0 software (IBM Company, Foster City, CA, USA). All experimental data were assessed using one-way analysis of variance (ANOVA), and results were presented as the means ± standard error (SE) at the levels of * p < 0.05, ** p < 0.01, and *** p < 0.001.

Conclusions
In brief, this study systematically analyzed the YUCCA gene family structures, phylogenetic characteristics, and expression patterns in I. indigotica. One of YUCCA gene family members, IiYUCCA6-1, could promote IAA accumulation and the upregulation of some auxin-responsive genes, which triggered a typical hyperauxin performance. Moreover, the contents of total chlorophyll and H 2 O 2 decreased and the expression of NbSAG12 was downregulated, which could delay the senescence process while improving the stress resistance ( Figure 9). This study provided a systematic and comprehensive analysis of the I. indigotica YUCCA gene family using phylogenetic analysis, gene expression analysis, and functional characterization by heterologous expression in tobacco. All the findings demonstrated the experimental basis for the identification of the YUCCA genes in I. indigotica, with an exploration of their function. Statistical analysis was performed with SPSS 22.0 software (IBM Company, California, USA). All experimental data were assessed using one-way analysis of variance (ANOVA), and results were presented as the means ± standard error (SE) at the levels of * p < 0.05, ** p < 0.01, and *** p < 0.001.

Conclusions
In brief, this study systematically analyzed the YUCCA gene family structures, phylogenetic characteristics, and expression patterns in I. indigotica. One of YUCCA gene family members, IiYUCCA6-1, could promote IAA accumulation and the upregulation of some auxin-responsive genes, which triggered a typical hyperauxin performance. Moreover, the contents of total chlorophyll and H2O2 decreased and the expression of NbSAG12 was downregulated, which could delay the senescence process while improving the stress resistance ( Figure 9). This study provided a systematic and comprehensive analysis of the I. indigotica YUCCA gene family using phylogenetic analysis, gene expression analysis, and functional characterization by heterologous expression in tobacco. All the findings demonstrated the experimental basis for the identification of the YUCCA genes in I. indigotica, with an exploration of their function.