Identiﬁcation and Expression Analysis of the PIN and AUX / LAX Gene Families in Ramie ( Boehmeria nivea L. Gaud)

: Auxin regulates diverse aspects of growth and development. Furthermore, polar auxin transport, which is mediated by the PIN-FORMED (PIN) and AUXIN1 / LIKE-AUX (AUX / LAX) proteins, plays a crucial role in auxin distribution. In this study, six PIN and four AUX / LAX genes were identiﬁed in ramie ( Boehmeria nivea L.). We used qRT-PCR to characterize and analyze the two gene families, including phylogenetic relationships, intron / exon structures, cis-elements, subcellular localization, and the expression patterns in di ﬀ erent tissues. The expression of these genes in response to indole-3-acetic acid (IAA) treatment and drought stress was also assessed; the results indicate that most of the BnAUX / LAX and BnPIN genes were regulated as a result of IAA treatment and drought stress. Our study provides insights into ramie auxin transporters and lays the foundation for further analysis of their biological functions in ramie ﬁber development and adaptation to environmental stresses. the BnAUX / LAX and BnPIN gene families. Gene identiﬁcation and structure, basic parameters, phylogenetics, promoter cis-regulatory element analysis, tissue expression patterns, transcriptional responses to hormone treatment and abiotic stress, and subcellular localization are addressed. The results of this study could provide a foundation for further research.


Introduction
Auxin is a phytohormone that controls numerous aspects of plant growth and developmental processes, including apical dominance [1], phloem and wood formation [2,3], flower abscission [4], fruit and root development [5,6], phototropism [7], and leaf formation [8]. In addition, auxin participates in plant responses to abiotic stresses [9,10]. Indole-3-acetic acid (IAA) is the main form of auxin in plant hormones. There are two distinct pathways of auxin transport in plants: passive transport through phloem and active intercellular transport. Auxin influx and efflux carriers promote the intercellular movement of auxin [11]. Polar auxin transport (PAT), combined with local auxin biosynthesis, plays an important role in maximizing auxin production, and is essential for plant development and stress responses [12,13]. The interaction and coordination of auxin influx and efflux carrier proteins in plants constitute a flexible network that can respond to environmental and developmental changes. The four known auxin transporter families in plants are the PIN family, PIN-LIKES (PILS) family, AUX/LAX family, and ATP-binding cassette family B (ABCB)-P-glycoprotein (PGP) family [14,15]. Among these four families, AUX/LAX and PIN are the most well-characterized families involved in auxin influx and auxin efflux.
AUX/LAX proteins transport auxin into cells [16]. In Arabidopsis thaliana, the AUX/LAX family consists of four highly conserved genes: AUX1, LAX1, LAX2, and LAX3 [17]. AUX/LAX genes encode multimembrane-spanning transmembrane proteins, and biochemical and genetic evidence suggests were cut, and the incisions were immersed in 0.1 g/L KMnO 4 for 2 days and then cultured in water for rooting. Afterward, all plants were transferred to Hoagland's nutrient solution for 7 days. Some plants were then treated with 0.05 M IAA (Sigma-Aldrich, Saint Louis, MO, USA), while others continued to grow in Hoagland. After 60 min of IAA treatment, the leaves were sampled, immediately frozen in liquid nitrogen, and stored at −80 • C. There were three biological replicates for each sample.

Identification of BnPIN and BnAUX/LAX Auxin Transporter Gene Families in Ramie
The AtPIN and AtAUX/LAX gene sequences were obtained from the TAIR database [55]. Because mulberry (Morus notabilis) and ramie both belong to the order Urticales, the MnPIN and MnAUX/LAX gene sequences were downloaded from the mulberry genome database [56]. All the obtained sequences of the two gene families were used to search three ramie transcriptome databases [57][58][59]. ClustalX [60] was used to align the sequences from the three transcriptome databases according to the nucleotide sequence. If two or more sequences from different databases overlapped partially (more than 50 bp) or completely, they were further assembled. Finally, all the aforementioned genes obtained were analyzed by using the Open Reading Frame Finder [61] to obtain the coding sequences (CDSs), which were submitted to GenBank [62] ( Table 1). The genome sequences of the BnPIN and BnAUX/LAX gene families were obtained using the CDSs to conduct a BLASTN search in the two ramie genome databases [54,63]. In this study, phylogenetic relationships were constructed with all the BnAUX/LAX and BnPIN amino acid sequences of Arabidopsis, mulberry, and ramie using the neighbor-joining (NJ) method in MEGA software (version 5.0), and the NJ tree was evaluated by 1000 bootstrap replicates [64]. Conserved functional domains in the protein sequences were analyzed by online MEME software (version 5.0.4) [65]. Protein transmembrane topology was predicted using TMHMM Server (version 2.0) [66]. The protein lengths, molecular weights, and theoretical isoelectric points were analyzed by the online ProtParam tool of ExPASy server [67]. Protein subcellular localization was predicted online by CELLO (version 2.5) [68].

Cis-Elements in the Promoter Regions of BnAUX/LAX and BnPIN Genes
The cis-elements in the BnPIN and BnAUX/LAX gene promoter regions were surveyed by searching the ramie genome database to retrieve 2 kb sequences that are upstream of the initiation codon. The putative cis-acting elements associated with stress responses, growth, and development were identified online by PlantCARE [69]. The image data were displayed using TBtools software (version 0.6652) [70].

RNA Extraction and Real-Time Quantitative PCR Analysis
RNA was extracted using the RNA Prep Pure Plant kit (Tiangen Biotech, Beijing, China) and then reverse-transcribed by the GoScript Reverse Transcription System (Promega, Madison, MI, USA). Quantitative real-time PCR was performed on a Bio-Rad iQ5 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). The glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene was selected as the internal control [58]. Specific primers were designed online (http://primer3.ut.ee/) ( Table S1). The 20 µL reaction system included 1 µL of cDNA, 1 µL of forward primers, 1 µL of reverse primers, 10 µL of iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA), and 7 µL of ddH 2 O. The thermocycling regime consisted of 5 min at 95 • C, 40 cycles of 15 s at 95 • C, and 30 s at 60 • C. Each sample was replicated three times. The data were calculated by the 2 −∆∆Ct method [71].

Subcellular Localization of BnAUX/LAX and BnPIN Proteins
To further confirm the subcellular localization of the BnPIN5 and BnAUX/LAX proteins, we constructed a BnPIN (AUX/LAX):GFP fusion gene controlled by the CaMV35S promoter (refer to Figure 7a). Specific primers were designed from both ends of the selected sequence (Table S2). Then, the fusion genes and empty vector (positive control) were transformed into tobacco (Nicotiana benthamiana) by Agrobacterium-mediated infiltration as described previously [72]. After transient transformation, the tobacco plants were grown in the dark for 24-48 h at room temperature, and then the epidermal cells were examined by a laser scanning confocal microscope (Olympus FV1200, Japan), the green fluorescence was excited with a 488-nm laser line, and cells were detected using a NIBA emission filter. The epidermal cells of untreated tobacco leaves were also examined as negative controls. The images were processed by Adobe PhotoshopCC2017.

Identification and Phylogenetic Analysis of Ramie BnAUX/LAX and BnPIN Families
In total, four BnAUX/LAX and six BnPIN genes were identified in ramie (Table S3). From the phylogenetic tree, the BnLAX gene family can be divided into two subfamilies ( Figure 1a). BnAUX1 and BnLAX1 belong to subfamily I, and BnLAX2 and BnLAX3 belong to subfamily II. A total of 20 PIN proteins, including 6 BnPIN, 8 AtPIN, and 6 MnPIN proteins, were used to construct a phylogenetic tree ( Figure 1b). The BnPIN family was divided into four subgroups. BnPIN3 belongs to subgroup I, BnPIN1a and BnPIN1b belong to subgroup II, BnPIN6 and BnPIN8 belong to subgroup III, and BnPIN5 belongs to subgroup IV. Moreover, most BnAUX/LAX and BnPIN proteins were more similar to those in mulberry compared with those in Arabidopsis.

Phylogenetic, Protein Domain, and Gene Structure Analysis of the BnAUX/LAX and BnPIN Families
The conserved motifs of the AUX/LAX and PIN proteins were investigated by MEME ( Figure 2). The AUX/LAX protein sequences have a conserved domain with a length of more than 400 amino acids

Cis-Element Prediction in BnAUX/LAX and BnPIN Promoters
Cis-acting elements that are bound by transcription factors and involved in plant stress response, growth, and development [73], among other processes. Promoter cis-element analysis reveals several phytohormone-related and stress-related motifs in the BnAUX/LAX and BnPIN gene promoter regions ( Figure 4, Table S4). Ten common cis-regulatory elements are briefly characterized as auxinresponsive, MeJA-responsive, salicylic acid-responsive, gibberellin-responsive, defense-and stressresponsive, abscisic acid-responsive, anaerobic-inducible, and drought-inducible elements. Furthermore, light-responsive elements are pervasive. These results indicate that the BnAUX/LAX and BnPIN genes are vital to various hormone signaling and abiotic stress responses, which might be hypothesized by their diverse natures.

Tissue-Specific and Treatment-Induced Expression Profiles of BnAUX/LAX and BnPIN
The tissue-specific expression levels of the BnAUX/LAX and BnPIN genes are shown in Figure  5. BnAUX1, BnLAX2, BnPIN3, BnPIN5, BnPIN6, and BnPIN8 were highly expressed in the leaves, while BnPIN1b had high expression levels in the bark. BnLAX1, BnPIN5, BnPIN6, and BnPIN8 were

Cis-Element Prediction in BnAUX/LAX and BnPIN Promoters
Cis-acting elements that are bound by transcription factors and involved in plant stress response, growth, and development [73], among other processes. Promoter cis-element analysis reveals several phytohormone-related and stress-related motifs in the BnAUX/LAX and BnPIN gene promoter regions ( Figure 4, Table S4). Ten common cis-regulatory elements are briefly characterized as auxin-responsive, MeJA-responsive, salicylic acid-responsive, gibberellin-responsive, defense-and stress-responsive, abscisic acid-responsive, anaerobic-inducible, and drought-inducible elements. Furthermore, light-responsive elements are pervasive. These results indicate that the BnAUX/LAX and BnPIN genes are vital to various hormone signaling and abiotic stress responses, which might be hypothesized by their diverse natures.

Tissue-Specific and Treatment-Induced Expression Profiles of BnAUX/LAX and BnPIN
The tissue-specific expression levels of the BnAUX/LAX and BnPIN genes are shown in Figure 5. BnAUX1, BnLAX2, BnPIN3, BnPIN5, BnPIN6, and BnPIN8 were highly expressed in the leaves, while BnPIN1b had high expression levels in the bark. BnLAX1, BnPIN5, BnPIN6, and BnPIN8 were expressed at relatively low levels in four tissues. After IAA treatment, the relative expression of BnAUX1, BnLAX1, BnPIN1b, BnPIN5, and BnPIN8 decreased. Conversely, the relative expression of BnLAX2, BnPIN3, and BnPIN6 increased, and there was no significant change in the relative expression of BnLAX3 and BnPIN1a ( Figure 5).
phytohormone-related and stress-related motifs in the BnAUX/LAX and BnPIN gene promoter regions ( Figure 4, Table S4). Ten common cis-regulatory elements are briefly characterized as auxinresponsive, MeJA-responsive, salicylic acid-responsive, gibberellin-responsive, defense-and stressresponsive, abscisic acid-responsive, anaerobic-inducible, and drought-inducible elements. Furthermore, light-responsive elements are pervasive. These results indicate that the BnAUX/LAX and BnPIN genes are vital to various hormone signaling and abiotic stress responses, which might be hypothesized by their diverse natures.

Tissue-Specific and Treatment-Induced Expression Profiles of BnAUX/LAX and BnPIN
The tissue-specific expression levels of the BnAUX/LAX and BnPIN genes are shown in Figure  5. BnAUX1, BnLAX2, BnPIN3, BnPIN5, BnPIN6, and BnPIN8 were highly expressed in the leaves, while BnPIN1b had high expression levels in the bark. BnLAX1, BnPIN5, BnPIN6, and BnPIN8 were expressed at relatively low levels in four tissues. After IAA treatment, the relative expression of BnAUX1, BnLAX1, BnPIN1b, BnPIN5, and BnPIN8 decreased. Conversely, the relative expression of BnLAX2, BnPIN3, and BnPIN6 increased, and there was no significant change in the relative expression of BnLAX3 and BnPIN1a ( Figure 5).  In bast fiber development, expression of the BnAUX1, BnLAX2, and BnPIN1a genes was inhibited. In the early stage of fiber development (the top part of the stem bark), only BnLAX3, BnPIN1b, and BnPIN3 were highly expressed; in the middle part of the bast fiber, the BnPIN5, BnPIN6, BnPIN8 genes were distinctly upregulated. The bottom part of the bast fiber represents the mature fiber, and the BnLAX3, BnPIN1b, and BnPIN3 genes were expressed to a higher degree relative to others. In vitro organogenesis includes the development of callus and shoot buds during regeneration, and intervals of 0 (W0), 4 (W1), 14 (W2), 28 (W3), and 35 (W4) days (the buds were observed for 30 days) were set on the basis of morphological observation. BnAUX1, BnPIN1a, and BnPIN1b were more expressed than other genes, and BnPIN6 was upregulated. Polyethylene glycol (PEG) treatment for 24 h caused the downregulation of the expression levels of most BnAUX/LAX and BnPIN genes, which were still downregulated after treatment for 72 h. In contrast, in the roots, most BnAUX/LAX and BnPIN genes were upregulated when treated for 24 h, and most genes were downregulated when PEG treatment lasted 24-72 h (Figure 6). It is worth mentioning that 0-24 and 24-72 h PEG treatment caused upregulation of the BnLAX1 gene in the roots for both periods, while the expression level of BnPIN5 was downregulated and then upregulated in the roots. (PEG) treatment for 24 h caused the downregulation of the expression levels of most BnAUX/LAX and BnPIN genes, which were still downregulated after treatment for 72 h. In contrast, in the roots, most BnAUX/LAX and BnPIN genes were upregulated when treated for 24 h, and most genes were downregulated when PEG treatment lasted 24-72 h (Figure 6). It is worth mentioning that 0-24 and 24-72 h PEG treatment caused upregulation of the BnLAX1 gene in the roots for both periods, while the expression level of BnPIN5 was downregulated and then upregulated in the roots.

Subcellular Localization of BnAUX/LAX and BnPIN5 Proteins
The positive control and the fusion constructs were transiently transformed into tobacco leaf cells. In Figure 7b, no GFP is observed in the negative control, and the GFP signal is distributed throughout the tobacco leaf cells in the GFP positive control. The GFP signals from the BnPIN5-GFP and BnAUX/LAX-GFP fusion proteins are observed clearly in the membrane, suggesting that the four fusion proteins were localized in the cell membrane.

Subcellular Localization of BnAUX/LAX and BnPIN5 Proteins
The positive control and the fusion constructs were transiently transformed into tobacco leaf cells. In Figure 7b, no GFP is observed in the negative control, and the GFP signal is distributed throughout the tobacco leaf cells in the GFP positive control. The GFP signals from the BnPIN5-GFP and BnAUX/LAX-GFP fusion proteins are observed clearly in the membrane, suggesting that the four fusion proteins were localized in the cell membrane.
(PEG) treatment for 24 h caused the downregulation of the expression levels of most BnAUX/LAX and BnPIN genes, which were still downregulated after treatment for 72 h. In contrast, in the roots, most BnAUX/LAX and BnPIN genes were upregulated when treated for 24 h, and most genes were downregulated when PEG treatment lasted 24-72 h (Figure 6). It is worth mentioning that 0-24 and 24-72 h PEG treatment caused upregulation of the BnLAX1 gene in the roots for both periods, while the expression level of BnPIN5 was downregulated and then upregulated in the roots.

Subcellular Localization of BnAUX/LAX and BnPIN5 Proteins
The positive control and the fusion constructs were transiently transformed into tobacco leaf cells. In Figure 7b, no GFP is observed in the negative control, and the GFP signal is distributed throughout the tobacco leaf cells in the GFP positive control. The GFP signals from the BnPIN5-GFP and BnAUX/LAX-GFP fusion proteins are observed clearly in the membrane, suggesting that the four fusion proteins were localized in the cell membrane.

Characterization and Analysis of BnAUX/LAX and BnPIN Genes in Ramie
Six PIN and four AUX/LAX genes were identified in ramie, a number of genes that is similar to the number in Arabidopsis. The biological functions of the AUX/LAX and PIN genes have been revealed in Arabidopsis. Therefore, studying the evolutionary relationships of AUX/LAX and PIN proteins among ramie, mulberry, and Arabidopsis can help us understand the possible biological functions of these genes. The phylogenetic analysis shows that the phylogenetic relationship between ramie and mulberry is closer than that between ramie and Arabidopsis. It is predicted that all BnAUX/LAX and BnPIN proteins are localized in the membrane, and the subcellular localization of the BnAUX1, BnLAX1, BnLAX2, and BnPIN5 proteins in tobacco are located in the membrane. The PIN5 protein has a reduced hydrophilic ring which is typically located in the internal compartment [40]. However, another study pointed out that the PIN5 protein is clearly localized in the PM [74]. For the BnAUX/LAX and BnPIN genes, we also explored the cis-regulatory elements in the promoter regions and discovered the enrichment of several hormone-and stress-related cis-elements, as well as many light-responsive elements (Table S4). The prediction of the cis-regulatory elements indicates that that BnAUX/LAX and BnPIN genes may participate in the drought stress response and drought tolerance. In Arabidopsis, PIN play important roles in regulating asymmetrical auxin translocation during phototropism [38]. Among them, PIN3 regulates the lateral translocation of auxin and plays a role in gravitropism and phototropism [38,75].

Analyses of Tissue-Specific Expression of BnAUX/LAX and BnPIN Genes
The differential expression of most BnAUX/LAX and BnPIN genes in tissues indicates that they may be involved in the regulation of ramie growth and development. Nearly all the BnAUX/LAX and BnPIN genes are highly expressed in the leaves. In Arabidopsis, the vein patterning in leaf is controlled by two distinct auxin transport pathways: PIN1-mediated intercellular auxin transport in the PM and PIN6-, PIN8-, and PIN5-mediated intracellular auxin transport in the endoplasmic reticulum [76]. Moreover, phyllotaxis changes when AUX1/LAX activity is lost [23]: the quadruple mutant aux1 lax1 lax2 lax3 and the single mutants aux1, lax2, and lax3 exhibit enhanced asymmetry in their venation patterns [20]. Therefore, we infer that the BnAUX/LAX and BnPIN genes may regulate auxin transport during leaf development. The BnLAX1, BnLAX2, BnPIN1a, BnPIN5, and BnPIN6 genes show low expression levels in the bark. In contrast, BnPIN1b is strongly expressed in the bark. In an analysis of PIN genes in cotton, fiber elongation was observed when the expression of PIN genes was increased [77]. Further research on BnPIN1b may increase our knowledge of the molecular mechanisms underlying bast fiber development in ramie.

BnAUX/LAX and BnPIN Genes Were Responsive to IAA Treatment and Drought Stress
Previous studies have reported crosstalk between auxin and biotic and abiotic stress signaling [78]. To confirm whether the BnAUX/LAX and BnPIN genes participate in IAA signaling and drought responses, we analyzed the gene expression levels in ramie treated with IAA and PEG. Many BnAUX/LAX and BnPIN genes responded to IAA treatment and drought stress at the transcriptional level, and they were differentially expressed in leaf and root in response to drought stress. In soybean, most of the PIN genes respond to a variety of phytohormone stimuli and abiotic stresses [79]. In sorghum, most of the SbPIN genes are upregulated by IAA treatment, and IAA induces SbLAX2 and SbLAX3, but the expression of SbLAX1 and SbLAX4 is inhibited in leaf and root [80]. In maize, the expression of most ZmPIN and ZmLAX genes is upregulated in the shoots, but these genes are downregulated in the roots as a result of drought stress [51]. In rice, the IAA content is reduced after drought stress. In response to these stresses, many genes involved in IAA biosynthesis and signaling change at the transcriptional level, and these changes are basically consistent with changes in the level of endogenous IAA [81]. OsPIN3t is involved in auxin transport and the drought stress response, suggesting that the polar auxin transport pathway is involved in regulating plant responses to water stress [82]. The synergistic or antagonistic hormone action and the coordinated regulation of hormone biosynthetic pathways play key roles in plant adaptation to abiotic stresses [83]. The versatile expression responses of BnAUX/LAX and BnPIN genes to IAA and drought stress suggest that these genes are controlled by complex regulatory networks. This is supported by the prediction analysis of the cis-element in the promoters of BnAUX/LAX and BnPIN. Drought stress severely affects ramie stem growth, and fiber production is easily affected by an arid environment [84]. AUX/LAX and PIN in ramie might promote plant adaptation to drought stress by participating in the regulation of auxin distribution.

Conclusions
This study comprehensively analyzed the AUX/LAX and PIN genes in ramie. Further research, such as the identification of biological functions and genetic analysis of each BnAUX/LAX and BnPIN gene, will accelerate the study of the molecular mechanisms mediated by auxin transporters that regulate fiber development and abiotic stress tolerance. The results of such studies can be used to increase the yields of ramie fiber and enhance the resistance to various stresses, thus improving plant performance.

Conflicts of Interest:
The authors declare no conflict of interest.