A Sweetpotato Auxin Response Factor Gene (IbARF5) Is Involved in Carotenoid Biosynthesis and Salt and Drought Tolerance in Transgenic Arabidopsis

Auxin response factors (ARFs) compose a family of transcription factors and have been found to play major roles in the process of plant growth and development. However, their roles in plant carotenoid biosynthesis and responses to abiotic stresses are rarely known to date. In the present study, we found that the IbARF5 gene from sweetpotato (Ipomoea batatas (L.) Lam.) line HVB-3 increased the contents of carotenoids and enhanced the tolerance to salt and drought in transgenic Arabidopsis. The transgenic Arabidopsis plants exhibited the increased abscisic acid (ABA) and proline contents and superoxide dismutase (SOD) activity and the decreased H2O2 content. Furthermore, it was found that IbARF5 positively regulated the genes associated with carotenoid and ABA biosynthesis and abiotic stress responses. These results suggest that IbARF5 is involved in carotenoid biosynthesis and salt and drought tolerance in transgenic Arabidopsis. This study provides a novel ARF gene for improving carotenoid contents and salt and drought tolerance of sweetpotato and other plants.


INTRODUCTION
In nature, more than 750 kinds of carotenoids are characterized structurally, which are widely found in bacteria, fungi, algae, and plants (Hirschberg, 2001;Takaichi, 2011). The biosynthesis pathway of carotenoids has been extensively studied in plants, and nearly all of the key genes have been isolated and characterized (Cunningham and Gantt, 1998;Fraser and Bramley, 2004;Colasuonno et al., 2017;Kang et al., 2018). Abiotic stresses, especially salt and drought, seriously affect the productivity and cultivation expansion of crop plants worldwide, accordingly, to develop their high tolerance to salt and drought is highly desirable (Zhu, 2002;Lindemose et al., 2013;Zhai et al., 2016;Li et al., 2017). As the precursor of abscisic acid (ABA), carotenoids have functional roles in development and environmental adaptation of plants (Schwartz et al., 2003;Nambara and Marion-Poll, 2005;Mehrotra et al., 2014;Li, 2015;Moreno et al., 2016). Thus, increasing the contents of carotenoids helps to enhance the adaptation of plants to harsh environments.
Auxin response factors (ARFs) constitute a family of plant specific transcription factors. A typical ARF protein contains a B3-DNA binding domain in the highly conserved N-terminal region (Ulmasov et al., 1997;Hagen and Guilfoyle, 2002;Mei et al., 2018). ARFs mediate responses to auxin and have been shown to be implicated in senescence , hormone signaling (Li et al., 2006) and developmental programs (Krogan et al., 2012). In rice, OsARF1 was auxin-regulated and classified as a primary auxin responsive gene (Waller et al., 2002). In Arabidopsis, ARF2 mediated ABA response (Wang et al., 2011); MP/ARF5 regulated embryo and flower patterning and vascular differentiation (Hardtke and Berleth, 1998;Krogan et al., 2012); ARF6 and ARF8 promoted jasmonic acid production and flower maturation ; NPH4/ARF7 and ARF19 controlled leaf expansion and lateral root growth (Okushima et al., 2005;Wilmoth et al., 2005). In tomato, SlARF2 regulated lateral root formation and flower senescence (Ren et al., 2017); ARF4 controlled sugar metabolism (Sagar et al., 2013); ARF10 increased chlorophyll and sugar accumulation during fruit development (Mei et al., 2018); ARF5 regulated fruit set and development . However, the roles of ARFs in plant carotenoid biosynthesis and abiotic stress responses are rarely known to date.
Sweetpotato (Ipomoea batatas (L.) Lam.) is an important food crop worldwide, which provides rich carbohydrates and carotenoids for human consumption (Teow et al., 2007;Zhai et al., 2016). This crop can also be used for bioenergy production on marginal lands due to its high adaption to harsh environments . Sweetpotato breeders are focusing on improving carotenoid contents and abiotic stresses tolerance of this crop. Kang et al. (2017) summarized the improvement of carotenoids by gene engineering in sweetpotato. Overexpression of the genes related to carotenoid biosynthesis have been shown to increase the contents of carotenoids and enhance the tolerance to abiotic stresses in sweetpotato (Kim et al., 2012, Kim et al., 2013bYu et al., 2013;Kim et al., 2014;Li et al., 2017;Kang et al., 2018). To date, ARFs have not been reported in sweetpotato. In this study, we found that the IbARF5 gene from storage roots of sweetpotato is involved in carotenoid biosynthesis and salt and drought tolerance in transgenic Arabidopsis.

Plant Materials
Sweetpotato line HVB-3 with high carotenoid content was employed to clone the IbARF5 gene in this study. The expressed sequence tag (EST) for IbARF5 was obtained from the transcriptome data of HVB-3 developed by Li et al. (2015). Arabidopsis wild type (Columbia-0, WT) was used for characterizing the IbARF5 gene.

Isolation and Sequence Analysis of IbARF5
Total RNA was extracted from freshly harvested storage roots of HVB-3 and transcribed into first-strand cDNA according to the method of Kang et al. (2018). The full-length cDNA of   IbARF5 was amplified with specific primers (Supplementary Table S1) by rapid amplification of cDNA ends (RACE) method. Genomic DNA isolated from in vitro-grown plants of HVB-3 was used to amplify the genomic sequence of IbARF5. The IbARF5 cDNA was analyzed by an online BLAST 1 . The openreading frame (ORF) Finder 2 was used to predict the ORF of IbARF5. The DNAMAN software was applied to align the amino acid sequence of IbARF5 with those of ARF proteins from different plant species. The MEGA 7.0 software was employed to conduct the phylogenetic analysis with the neighbor-joining (NJ) method. Exon-intron structure was constructed using Splign tool 3 . The molecular weight and theoretical isoelectric point (pI) of IbARF5 were calculated at http://web.expasy.org/compute_ pi/.

Subcellular Localization of IbARF5
The IbARF5 ORF amplified with specific primers (Supplementary Table S1) was ligated into pMDC83. pMDC83-IbARF5-GFP and pMDC83-GFP (as control) were transiently expressed in the onion epidermal cells with a GeneGun (HeliosTM, Biorad, United States). After co-cultivation on Murashige and Skoog (MS) medium (pH 5.8) at 28 • C for 24 h, the onion cells were examined under a laser scanning confocal fluorescence microscope (Nikon Inc., Melville, NY, United States).

Expression Analysis of IbARF5 in Sweetpotato
Total RNA was isolated from storage root, stem, and leaf tissues of the 100-day-old HVB-3 and used to analyze the expression of IbARF5 by quantitative real-time PCR (qRT-PCR) with its specific primers (Supplementary Table S1). Ibactin (AY905538) was served as an internal control. Comparative C T method was employed to quantify the gene expression (Schmittgen and Livak, 2008).
After cultured on MS medium for 4 weeks, the HVB-3 plants were treated in liquid MS media containing H 2 O (as control), 200 mM NaCl, 20% PEG6000 and 100 µM ABA, respectively, and

Production of the Transgenic Arabidopsis Plants
The overexpression vector pC3301-121-IbARF5 was constructed through inserting 35S-IbARF5-NOS into pCAMBIA3301. The recombinant vector was introduced into the Agrobacterium tumefaciens strain GV3101. The dipping flower method was applied to transform Arabidopsis and putatively transgenic Arabidopsis seeds were sown on MS medium with 12.5 mg L −1 phosphinothricin (PPT) for selecting transgenic plants. Histochemical GUS assay (Jefferson et al., 1987) and PCR analysis were used to identify the transgenic Arabidopsis plants. Transgenic Arabidopsis was planted in pots with a soil, vermiculite and humus mixture (1:1:1, v/v/v) to obtain T 3 seeds.

Measurement of Carotenoid Contents
Leaves (2-week-old) and seeds of the transgenic Arabidopsis plants were applied to extract α-carotene, lutein, β-carotene, β-cryptoxanthin, and zeaxanthin. High performance liquid chromatography (HPLC) system was used to determine their contents (Li et al., 2017).

Assay for Salt and Drought Tolerance
One-week-old in vitro-grown seedlings of transgenic Arabidopsis and WT were treated on MS media with 200 mM NaCl and 300 mM mannitol, respectively. After 2 weeks, their root length and fresh weight (FW) were investigated. Furthermore, the transgenic and WT seedlings were planted for 2 weeks in pots with a soil, vermiculite and humus mixture (1:1:1, v/v/v) and subsequently irrigated with a 33 mL of 300 mM NaCl solution for each pot once every 2 days for 2 weeks, or stressed by drought for 4 weeks followed by 2 days re-watering. The transgenic plants and WT grown for 6 weeks under normal condition were used as control. The proline and H 2 O 2 contents and superoxide dismutase (SOD) activity in the transgenic plants and WT grown in pots for 4 weeks under normal condition, 1 week under 300 mM NaCl stress after 2 weeks of normal treatment, FIGURE 4 | Expression analysis of IbARF5 in storage root, stem, and leaf tissues of HVB-3. Data are presented as means ± SE (n = 3). Different capital letters indicate a significant difference at P < 0.01 by Student's t-test.
Frontiers in Plant Science | www.frontiersin.org and 2 weeks under drought stress after 2 weeks of normal treatment, respectively, were determined with Assay Kits (Comin Biotechnology Co., Ltd. Suzhou, China). The ABA content was measured as described by Gao et al. (2011). Twenty-seven plants in three pots with nine plants per pot were treated for each line.
For ABA sensitivity assay, the transgenic and WT seeds were sown on MS media with 0, 0.5, and 1 µM ABA, respectively. After 1 week, their germination and cotyledon opening and greening rates were investigated. Fifty seeds of each line on a plate were analyzed.

Expression Analysis of the Related Genes
Leaves (2-week-old) and seeds of the transgenic Arabidopsis plants and WT were applied to analyze the expression of the key genes in carotenoid biosynthesis. The leaves of the transgenic plants and WT potted for 4 weeks under normal condition, 1 week under 300 mM NaCl stress after 2 weeks of normal treatment, and 2 weeks under drought stress after 2 weeks of normal treatment, respectively, were used for analyzing the expression of the genes associated with ABA biosynthesis and abiotic stress responses. The specific primers of Atactin FIGURE 5 | Expression analysis of IbARF5 in the in vitro-grown plants of HVB-3 after different times (h) in response to H 2 O, 200 mM NaCl, 20% PEG6000 and 100 µM ABA, respectively. Data are presented as means ± SE (n = 3). * and * * indicate a significant difference from that of WT at P < 0.05 and P < 0.01, respectively, by Student's t-test.

Statistical Analysis
All experiments were conducted with three biological replicates. Data presented as the mean ± SE were analyzed with Student's t-test (two-tailed analysis) at P < 0.05 and P < 0.01.

Cloning and Sequence Analysis of IbARF5
The 3757 Figure S1). Phylogenetic analysis showed that IbARF5 had a close relationship with that of N. tabacum ( Figure 1A). The 4794-bp genomic DNA of IbARF5 contained 13 exons and 12 introns ( Figure 1B).

IbARF5 Is Localized to Nuclei
The images from onion epidermal cells indicated that the green fluorescence emitted by IbARF5-GFP was exclusively distributed over the nuclei of the cells (Figure 2). These results showed that IbARF5 was localized to nuclei.

IbARF5 Shows Transactivation Activity in Yeast
The yeast two-hybrid system was applied to identify a possible transactivation activity of IbARF5. The yeast cells harboring pBD-GAL4, pGBKT7-IbARF5 and pGBKT7-IbARF5-2, respectively, grew well on synthetic dropout (SD) plate without tryptophan and histidine and exhibited β-galactosidase activity, but the cells bearing pBD, pGBKT7-IbARF5-1, and pGBKT7-IbARF5-3, respectively, failed to grow ( Figure 3A). These results demonstrated that IbARF5 might act as a transcription activator, and its transactivation activity was determined by the middle region, IbARF5-2 ( Figure 3B).
FIGURE 6 | Responses of the transgenic Arabidopsis seedlings and WT cultured for 2 weeks on MS medium with 200 mM NaCl and 300 mM mannitol, respectively. Data are presented as mean ± SE (n = 3). * * indicates a significant difference from that of WT at P < 0.01 by Student's t-test. week under 300 mM NaCl stress after 2 weeks of normal treatment, and 2 weeks under drought stress after 2 weeks of normal treatment, respectively. Data are presented as mean ± SE (n = 3). * * indicates a significant difference from that of WT at P < 0.01 by Student's t-test.
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Expression Patterns of IbARF5 in Sweetpotato
Quantitative real-time PCR analysis revealed that IbARF5 exhibited higher expression level in the storage roots of HVB-3 than in its leaves and stems (Figure 4). Its expression in HVB-3 was strongly induced by NaCl, PEG6000 and ABA, and peaked (5.03-fold) at 4 h, (9.68-fold) at 24 h, and (12.18-fold) at 24 h, respectively (Figure 5).

Production of the Transgenic Arabidopsis Plants
Putatively transgenic Arabidopsis seeds formed the plants on MS medium with 12.5 mg L −1 PPT. GUS assay and PCR analysis confirmed that 6 of the randomly sampled 60 plants were transgenic plants, named L1, L2, . . ., L6, respectively, from which T 3 were generated. IbARF5 showed high expression levels in the transgenic Arabidopsis plants, especially L1, L4, L5, and L6 (Supplementary Figure S2).

IbARF5 Increases Carotenoid Contents
The lutein and zeaxanthin contents were significantly increased, but α-carotene and β-carotene contents were not changed and β-cryptoxanthin was not detected in leaves of L1, L4, L5, and L6 (Table 1). In seeds of these four transgenic plants, the lutein, β-carotene, and zeaxanthin contents were significantly increased, but α-carotene was not detected and β-cryptoxanthin content was significantly increased only in L5 and L6 ( Table 2). The total carotenoid contents in leaves and seeds were increased by 1.13-1.16 folds and 1.32-1.78 folds, respectively (Tables 1 and 2).

IbARF5 Enhances Salt and Drought Tolerance
Four transgenic Arabidopsis plants, L1, L4, L5, and L6, and WT seedlings showed no significant differences in rooting and FW on MS medium without stresses (Figure 6). However, the transgenic plants exhibited good rooting and increased FW in contrast to WT on MS media with 200 mM NaCl and 300 mM mannitol, respectively (Figure 6). The transgenic plants and WT grown in pots showed similar growth trends under normal conditions ( Figure 7A). Under NaCl and drought stresses, the transgenic plants showed good growth, while WT almost died ( Figure 7A). Furthermore, it was found that the ABA and proline contents were increased, SOD activity Frontiers in Plant Science | www.frontiersin.org was enhanced and H 2 O 2 content was decreased in the transgenic plants ( Figure 7B).
No obvious differences in seed germination were observed between the transgenic plants and WT under normal condition (Figure 8). Under the treatment with different ABA concentrations, the germination of the transgenic seeds was more sensitive to ABA-elicited inhibition than that of WT though both germination rate and cotyledon opening and greening rate of the transgenic and WT seeds declined (Figure 8). These results demonstrated that IbARF5 might participate in the ABA signaling pathway.

IbARF5 Increases Carotenoid Contents and Salt and Drought Tolerance
In plants, ARFs encode important transcription factors which regulate the expression of genes in response to auxin (Guilfoyle and Hagen, 2007). Several ARF transcription factor genes have FIGURE 9 | Transcript levels of IbARF5 and carotenoid biosynthesis genes in leaves and seeds of the transgenic Arabidopsis plants and WT. Data are presented as means ± SE (n = 3). * and * * indicate a significant difference from that of WT at P < 0.05 and P < 0.01, respectively, by Student's t-test. been cloned from Arabidopsis, rice and tomato, and were found to play crucial roles in plant growth and developmental processes (Harper et al., 2000;Waller et al., 2002;Ellis et al., 2005;Okushima et al., 2005;Wilmoth et al., 2005;Wang et al., 2011;Ren et al., 2017;Liu et al., 2018). However, there is no report on the ARF transcription factors in improving carotenoid contents and abiotic stress tolerance in plants. Previous studies demonstrated that AtARF5 affected lateral organ development, primary root initiation, flower primordium initiation, and cotyledon development in Arabidopsis (Krogan et al., 2012) and SlARF5 controlled fruit set and development in tomato . In the present study, the IbARF5 gene was isolated from sweetpotato line HVB-3 with high carotenoid content. We found that its overexpression significantly increased the content of carotenoids and enhanced the tolerance to salt and drought in transgenic Arabidopsis (Tables 1, 2 and Figures 6, 7).
FIGURE 10 | Transcript levels of salt and drought responsive genes in leaves of transgenic Arabidopsis plants and WT pot-grown for 4 weeks under normal condition, 1 week under 300 mM NaCl stress after 2 weeks of normal treatment, and 2 weeks under drought stress after 2 weeks of normal treatment, respectively. Data are presented as mean ± SE (n = 3). * * indicates a significant difference from that of WT at P < 0.01 by Student's t-test.
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IbARF5 Up-Regulates the Genes Involved in Carotenoid Biosynthesis
It has been shown that carotenoid biosynthesis is mainly regulated at the transcript level of genes encoding the biosynthetic enzymes (Römer and Fraser, 2005;Sandmann et al., 2006;Li et al., 2017;Kang et al., 2018). In this study, we found that the key genes in carotenoid biosynthesis, GGPS, ZDS, PSY, ε-CHY, β-LCY, and β-CHY in leaves and GGPS, ε-CHY, β-LCY, and β-CHY in seeds of transgenic Arabidopsis were significantly up-regulated (Figure 9), which corresponded with the increase of carotenoid contents in transgenic Arabidopsis (Tables 1 and  2). These findings suggest that IbARF5 positively controls the expression of carotenoid biosynthetic genes, which resulted in the increased carotenoid contents in transgenic Arabidopsis. Overexpression of the Orange gene (IbOr) from sweetpotato increased carotenoid accumulation and abiotic stress tolerance in transgenic sweetpotato, potato, and alfalfa (Kim et al., 2013a;Goo et al., 2015;Wang et al., 2015). Furthermore, it was proved that similar to AtOr of Arabidopsis, IbOr directly interacted with PSY and increased carotenoid accumulation (Park et al., 2016;Kim et al., 2018). Therefore, the precise underlying mechanisms of IbARF5 in plant carotenoid accumulation need to be further investigated. In addition, we are developing the IbARF5-overexpressing sweetpotato plants for further analyzing its roles in carotenoid accumulation of the storage roots.

IbARF5 Up-Regulates the Genes Involved in ABA Biosynthesis
Carotenoids, especially β-branch carotenoids, serve as precursors for ABA biosynthesis and play a crucial role in plant tolerance and adaptation to abiotic stresses (Demmigadams and Adams, 2002;Xiong and Zhu, 2003;Sah et al., 2016). ABA regulates the expression of ABA-dependent stress-responsive genes and the increased level of ABA has been found to enhance the tolerance to salt and drought (Tuteja, 2007;Vishwakarma et al., 2017). It was reported that overexpression of IbMIPS1, IbZDS, and IbLCYB2 increased the level of ABA, which led to the enhanced tolerance to salt and drought in sweetpotato (Zhai et al., 2016;Li et al., 2017;Kang et al., 2018). In this study, the IbARF5overexpressing Arabidopsis seeds showed the increased sensitivity to ABA in germination (Figure 8). The ABA biosynthetic genes IbZEP, IbNCED, and IbABA2 were up-regulated and ABA level was also significantly increased in transgenic Arabidopsis (Figures 7B and 10). These results suggest that overexpression of IbARF5 confers salt and drought tolerance by up-regulating the ABA biosynthetic genes and increasing ABA level in transgenic Arabidopsis.

IbARF5 Up-Regulates Abiotic Stress-Responsive Genes and Changes Abiotic Stress-Associated Components
It is reported that the high level of ABA increases the transcript level of P5CS, which leads to more accumulation of proline under abiotic stresses (Sripinyowanich et al., 2013). Proline plays a pivotal role in maintaining osmotic balance, protecting integrity membrane and increasing reactive oxygen species (ROS) scavenging capacity, and its elevated level enhances salt and drought tolerance in plants (Yoshiba et al., 1997;Maggio et al., 2002;Neisiani et al., 2009;Gill and Tuteja, 2010;Kang et al., 2018). SOD as the first line of defense against ROS is induced by abiotic stresses to promote ROS scavenging (Wang et al., 2009). In the present study, P5CS, SOD, APX, and DHAR were up-regulated, proline level and SOD activity were increased and H 2 O 2 content was decreased in transgenic Arabidopsis under salt and drought stresses ( Figure 7B). Therefore, it is thought that the enhanced tolerance to salt and drought is due to upregulation of abiotic stress-responsive genes and change of abiotic stress-associated components in transgenic Arabidopsis.

CONCLUSION
This study reveals, for the first time, that the IbARF5 gene from sweetpotato is involved in carotenoid biosynthesis and salt and drought tolerance of plants. Its overexpression increased the contents of carotenoids and conferred the tolerance to salt and drought by up-regulating the key genes involved in carotenoid and ABA biosynthesis and abiotic stress responses in transgenic Arabidopsis. This study provides a novel ARF gene for improving carotenoid contents and salt and drought tolerance of sweetpotato and other plants.

AUTHOR CONTRIBUTIONS
QL and CK conceived and designed the experiments. CK and RL performed the experiments. CK and SH analyzed the data. QL, HZ, and NZ contributed reagents, materials, and analysis tools. QL and CK wrote the paper. All authors read and approved the final manuscript.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.01307/ full#supplementary-material FIGURE S1 | Sequence alignment of IbARF5 with its homologs from other plants. Characteristic regions of ARF5 are indicated above the IbARF5 sequence.
FIGURE S2 | Expression analysis of IbARF5 in the transgenic Arabidopsis plants.
The Arabidopsis actin gene was used as an internal control. Data are presented as means ± SE (n = 3). * * indicates a significant difference from that of WT at P < 0.01 by Student's t-test.