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Article

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

1
MOA Key Laboratory of Crop Ecophysiology and Farming System in the Middle Reaches of the Yangtze River, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
2
Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
*
Authors to whom correspondence should be addressed.
Submission received: 28 June 2019 / Revised: 26 July 2019 / Accepted: 3 August 2019 / Published: 7 August 2019
(This article belongs to the Special Issue Hormone Signaling and Regulation in Cultivated Plants)

Abstract

:
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 different 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.

1. 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 that members of the AUX/LAX family are functional auxin influx carriers and mediate auxin-related developmental programs in different tissues and organs [18]. From early embryonic development, cellular patterning requires AUX1/LAX-dependent auxin influx, and the expression of AUX1 and LAX2 is controlled by the MONOPTEROS-BODENLOS (MP-BDL) signaling pathway [16]. AUX1 is expressed in tissues related to gravity perception, signal transmission, and signal response [18,19], while LAX2 plays an important role in normal xylem development [20]. In addition, aux1 and lax1 mutants had significantly altered leaf phyllotaxy [21], lax1 and lax3 mutants had reduced lateral root formation [20,22], and the lax2 mutant had vascular breaks in cotyledons [17].
The auxin efflux carriers in the PIN family transport auxin throughout the plant in a polar manner [23,24,25]. PIN proteins are usually located in the plasma membrane (PM) or endoplasmic reticulum (ER) to direct the auxin flow. The first PIN family member was identified in Arabidopsis in association with auxin transport [26]. To date, eight PIN protein family members, named PIN1–PIN8, have been isolated from Arabidopsis [27]. Among them, the PIN1–4 and PIN7 proteins are localized in the PM and function as auxin efflux carriers. The PIN5, PIN6, and PIN8 proteins have a reduced hydrophilic loop in the middle which may regulate the auxin exchange between the cytosol and ER. Variations in the activity of these genes cause altered levels of free IAA and IAA conjugates and affect nuclear auxin signaling [13]. PINs have functional redundancy, and their biochemical activity is regulated in multiple stages [27]. Furthermore, the fewest PIN genes are found in Marchantia polymorpha, which has 4, while the most are found in Glycine max, which has 23 [28]. In Arabidopsis, PIN1 is involved in floral bud [26] and leaf shape formation [29], shoot vascular development [30], gravitropic and phototropic responses [31,32], and vein patterning [29]. PIN2 is expressed in cortical and epidermal cells of apical elongation zones [33,34] and is involved in the root gravitropic response [35]. PIN3 is involved in lateral root formation [36], apical hook formation and maintenance [37], phototropic responses [32], and gravitropism [38]. PIN4 plays roles in phototropic responses and apical hook development [35], and is expressed in the meristems of roots [39]. PIN5 is involved in early embryogenesis, cotyledon expansion, lateral root initiation, and root and hypocotyl growth [40]. PIN6 is dually localized in the PM and ER. It regulates intracellular auxin homeostasis and auxin transport during plant growth, including shoot apical dominance, adventitious root formation, root waving, root hair growth, and lateral root primordia development [41,42], and also participates in inflorescence stem elongation [43], production of nectar, and short stamen development [23]. PIN7 participates in gravitropic and phototropic responses [32,44], early embryogenesis [25], and apical hook development [45]. PIN8 acts as a pollen-specific auxin carrier, and is involved in sporophyte and male gametophyte development [28,46,47].
Although extensive research has been conducted on the AUX/LAX and PIN gene families in species throughout the plant kingdom, including Arabidopsis, Populus, Glycine max, Sorghum bicolor, Zea mays, Capsicum annuum, and cotton (Gossypium hirsutum) [47,48,49,50,51,52,53], little is known about these genes in ramie. Ramie has been cultivated for more than 4,700 years in China. The ramie fiber made from stem bast is an excellent textile material that is widely used in industrial fabrics and the manufacture of garments. Moreover, ramie is used as a forage crop in the south of China [54]. The present study provides comprehensive information about 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.

2. Materials and Methods

2.1. Plant Materials, Treatments, and Sampling

Ramie cv. 1504 was planted in the ramie germplasm repository of the Huazhong Agricultural University (Wuhan, Hubei Province, China). The shoots, leaves, stem bark, and roots of 2-month-old plants were sampled. For indole acetic acid (IAA) treatment, the tips of young shoots (about 15 cm) were cut, and the incisions were immersed in 0.1 g/L KMnO4 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.

2.2. 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].

2.3. Phylogenetic Analysis, Gene Structure, and Protein Profile Analysis

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].

2.4. 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].

2.5. 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 ddH2O. 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].

2.6. Expression Profiling of BnAUX/LAX and BnPIN Genes

To further investigate the BnAUX/LAX and BnPIN expression, RNA-Seq data (measured by normalized FPKM) including bast fiber development (top, middle, and bottom) [57], in vitro organogenesis (W0, W1, W2, W3, W4) [58], and drought stress (1, 24, 72 h) [59] was used for analysis. These data were presented in heat maps using the R software (version 3.6.0).

2.7. 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.

3. Results

3.1. 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.

3.2. 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 (Figure 2a). Ramie PIN proteins have two conserved domains of 170 and 161 amino acids, respectively (Figure 2b). The CDS of BnAUX/LAX varies between 1410 and 1491 bp, coding 469–496 amino acids; the molecular weight is 53098.19–55944.47, and the isoelectric point (pI) is 8.41–9.19. TMHMM2 software predicted 10 transmembrane helices in BnAUX/LAX proteins. The CDS of BnPIN varies between 1086 and 2022 and encodes 359–673 amino acids; the molecular weight is 39279.19–72588.55, and the pI is 6.30–9.40. The transmembrane helices of BnPIN proteins range from 8 to 10 (Table 1, Figure 3), and all PINs contain two hydrophobic domains separated by a hydrophilic loop. There are 6, 6, and 8 exons in BnLAX1, BnLAX2, and BnLAX3, respectively, and 7, 7, 6, 7, 6, and 4 exons in the BnPIN genes. The difference between the longest gene BnPIN6 (with a gene size of 12.9 kb) and the shortest gene BnPIN8 (1.9 kb) is mainly due to the total intron length (Figure 4).

3.3. 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.

3.4. 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.

3.5. 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.

4. Discussion

4.1. 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].

4.2. 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.

4.3. 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.

5. 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.

Supplementary Materials

The following are available online at https://0-www-mdpi-com.brum.beds.ac.uk/2073-4395/9/8/435/s1, Table S1: Primers for qRT-PCR analysis; Table S2: Primers for subcellular localization; Table S3: Gene sequences of BnAUX/LAX and BnPIN from three databases; Table S4: Cis-Element Prediction of BnAUX/LAX and BnPIN Promoters.

Author Contributions

Conceptualization, B.W. and X.H.; Formal analysis; Y.B. and X.H.; writing—original draft preparation, Y.B.; writing—review and editing, B.W., M.R., and Y.W.; supervision, D.P. and B.W.; project administration, D.P.; funding acquisition, D.P. and B.W.

Funding

This research was funded by the National Key Research and Development Project of China (2018YFD0201100) and National Natural Science Foundation of China (31171594, 31671736).

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Booker, J. Auxin Acts in Xylem-Associated or Medullary Cells to Mediate Apical Dominance. Plant Cell Online 2003, 15, 495–507. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Rodriguez, V.A. Wiring a plant: Genetic networks for phloem formation in Arabidopsis thaliana roots. New Phytol. 2015, 210, 45–50. [Google Scholar] [CrossRef] [PubMed]
  3. Brackmann, K.; Qi, J.; Gebert, M.; Jouannet, V.; Schlamp, T.; Grünwald, K.; Wallner, E.S.; Novikova, D.D.; Levitsky, V.G.; Agustí, J.; et al. Spatial specificity of auxin responses coordinates wood formation. Nat. Commun. 2018, 9. [Google Scholar] [CrossRef]
  4. Guan, X.; Xu, T.; Gao, S.; Qi, M.; Wang, Y.; Liu, X.; Li, T. Temporal and Spatial Distribution of Auxin Response Factor Genes During Tomato Flower Abscission. J. Plant Growth Regul. 2013, 33, 317–327. [Google Scholar] [CrossRef]
  5. Blilou, I.; Xu, J.; Wildwater, M.; Willemsen, V.; Paponov, I.; Friml, J.; Heidstra, R.; Aida, M.; Palme, K.; Scheres, B. The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots. Nature 2005, 433, 39–44. [Google Scholar] [CrossRef] [PubMed]
  6. Meng, L.; Song, W.; Liu, S.; Dong, J.; Zhang, Y.; Wang, C.; Xu, Y.; Wang, S. Light Quality Regulates Lateral Root Development in Tobacco Seedlings by Shifting Auxin Distributions. J. Plant Growth Regul. 2015, 34, 574–583. [Google Scholar] [CrossRef]
  7. Goyal, A.; Karayekov, E.; Galvão, V.C.; Ren, H.; Casal, J.J.; Fankhauser, C. Shade Promotes Phototropism through Phytochrome B-Controlled Auxin Production. Curr. Biol. 2016, 26, 3280–3287. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  8. Chen, S.H.; Zhou, L.J.; Xu, P.; Xue, H.W. SPOC domain-containing protein Leaf inclination3 interacts with LIP1 to regulate rice leaf inclination through auxin signaling. PLoS Genet. 2018, 14, e1007829-19. [Google Scholar] [CrossRef] [PubMed]
  9. Shani, E.; Salehin, M.; Zhang, Y.; Sanchez, S.E.; Doherty, C.; Wang, R.; Mangado, C.C.; Song, L.; Tal, I.; Pisanty, O.; et al. Plant Stress Tolerance Requires Auxin-Sensitive Aux/IAA Transcriptional Repressors. Curr. Biol. 2017, 27, 437–444. [Google Scholar] [CrossRef]
  10. Ding, Y.; Ma, Y.; Liu, N.; Xu, J.; Hu, Q.; Li, Y.; Wu, Y.; Xie, S.; Zhu, L.; Min, L.; et al. MicroRNAs involved in auxin signalling modulate male sterility under high-temperature stress in cotton (Gossypium hirsutum). Plant J. 2017, 91, 977–994. [Google Scholar] [CrossRef]
  11. Finet, C.; Jaillais, Y. AUXOLOGY: When auxin meets plant evo-devo. Dev. Biol. 2012, 369. [Google Scholar] [CrossRef] [PubMed]
  12. Casanova-Sáez, R.; Voss, U. Auxin Metabolism Controls Developmental Decisions in Land Plants. Trends Plant Sci. 2019. [Google Scholar] [CrossRef] [PubMed]
  13. Brumos, J.; Robles, L.M.; Yun, J.; Vu, T.C.; Jackson, S.; Alonso, J.M.; Stepanova, A.N. Local Auxin Biosynthesis Is a Key Regulator of Plant Development. Dev. Cell 2018, 47, 306–318.e5. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. Balzan, S.; Johal, G.S.; Carraro, N. The role of auxin transporters in monocots development. Front. Plant Sci. 2014, 5, 393. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  15. Grones, P.; Friml, J. Auxin transporters and binding proteins at a glance. J. Cell Sci. 2015, 128, 1–7. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Hélène, S.R.; Grunewald, W.; Sauer, M.; Cannoot, B.; Soriano, M.; Swarup, R.; Weijers, D.; Bennett, M.; Boutilier, K.; Friml, J. Plant embryogenesis requires AUX/LAX-mediated auxin influx. Development 2015, 142, 702–711. [Google Scholar]
  17. Péret, B.; Swarup, K.; Ferguson, A.; Seth, M.; Yang, Y.; Dhondt, S.; James, N.; Casimiro, I.; Perry, P.; Syed, A.; et al. AUX/LAX Genes Encode a Family of Auxin Influx Transporters That Perform Distinct Functions during Arabidopsis Development. Plant Cell 2012, 24, 2874–2885. [Google Scholar] [CrossRef]
  18. Swarup, R.; Péret, B. AUX/LAX family of auxin influx carriers—An overview. Front. Plant Sci. 2012, 3. [Google Scholar] [CrossRef]
  19. Swarup, R.; Friml, J.; Marchant, A.; Ljung, K.; Sandberg, G.; Palme, K.; Bennett, M. Localization of the auxin permease AUX1 suggests two functionally distinct hormone transport pathways operate in the Arabidopsis root apex. Genes Dev. 2001, 15, 2648–2653. [Google Scholar] [CrossRef]
  20. Moreno, P.; Guillermo, S.; Moreno, J.E.; Cabello, J.V.; Arce, A.L.; Otegui, M.E.; Chan, R.L. A role for LAX2 in regulating xylem development and lateral-vein symmetry in the leaf. Ann. Bot. 2017, 120, 577–590. [Google Scholar] [CrossRef]
  21. Bainbridge, K.; Guyomarc’h, S.; Bayer, E.; Swarup, R.; Bennett, M.; Mandel, T.; Kuhlemeier, C. Auxin influx carriers stabilize phyllotactic patterning. Genes Dev. 2008, 22, 810–823. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Swarup, K.; Benkova, E.; Swarup, R.; Casimiro, I.; Péret, B.; Yang, Y.; Parry, G.; Nielsen, E.; De Smet, I.; Vanneste, S.; et al. The auxin influx carrier LAX3 promotes lateral root emergence. Nat. Cell Biol. 2008, 10, 946–954. [Google Scholar] [CrossRef] [PubMed]
  23. Zazímalová, E.; Murphy, A.S.; Yang, H.; Hoyerova, K.; Hosek, P. Auxin Transporters—Why So Many? Cold Spring Harb. Perspect. Biol. 2010, 2, a001552. [Google Scholar] [CrossRef] [PubMed]
  24. Ljung, K. Auxin metabolism and homeostasis during plant development. Development 2013, 140, 943–950. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Adamowski, M.; Friml, J. PIN-Dependent Auxin Transport: Action, Regulation, and Evolution. Plant Cell 2015, 27, 20–32. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Okada, K.; Ueda, J.; Komaki, M.K.; Bell, C.J.; Shimura, Y. Requirement of the auxin polar transport system in early stages of Arabidopsis floral bud formation. Plant Cell 1991, 3, 677–684. [Google Scholar] [CrossRef] [PubMed]
  27. Zazímalová, E.; Křeček, P.; Skůpa, P.; Hoyerova, K.; Petrasek, J. Polar transport of the plant hormone auxin—The role of PIN-FORMED (PIN) proteins. Cell. Mol. Life Sci. 2007, 64, 1621–1637. [Google Scholar] [CrossRef]
  28. Zhou, J.J.; Luo, J. The PIN-FORMED Auxin Efflux Carriers in Plants. Int. J. Mol. Sci. 2018, 19, 2759. [Google Scholar] [CrossRef]
  29. Pahari, S.; Cormark, R.D.; Blackshaw, M.T.; Liu, C.; Erickson, J.L.; Schultz, E.A. Arabidopsis UNHINGED encodes a VPS51 homolog and reveals a role for the GARP complex in leaf shape and vein patterning. Development 2014, 141, 1894–1905. [Google Scholar] [CrossRef] [PubMed]
  30. Galweiler, L.; Guan, C.; Muller, A.; Wisman, E.; Mendgen, K.; Yephremov, A.; Palme, K. Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue. Science 1998, 282, 2226–2230. [Google Scholar] [CrossRef] [PubMed]
  31. Xi, W.; Gong, X.; Yang, Q.; Yu, H.; Liou, Y.C. Pin1At regulates PIN1 polar localization and root gravitropism. Nat. Commun. 2016, 7. [Google Scholar] [CrossRef] [PubMed]
  32. Haga, K.; Sakai, T. PIN auxin efflux carriers are necessary for pulse-induced but not continuous light-induced phototropism in Arabidopsis. Plant Physiol. 2012, 160, 763–776. [Google Scholar] [CrossRef] [PubMed]
  33. Laxmi, A.; Pan, J.; Morsy, M.; Chen, R. Light Plays an Essential Role in Intracellular Distribution of Auxin Efflux Carrier PIN2 in Arabidopsis thaliana. PLoS ONE 2008, 3, e1510-11. [Google Scholar] [CrossRef] [PubMed]
  34. Rahman, A.; Takahashi, M.; Shibasaki, K.; Wu, S.; Inaba, T.; Tsurumi, S.; Baskin, T.I. Gravitropism of Arabidopsis thaliana roots requires the polarization of PIN2 toward the root tip in meristematic cortical cells. Plant Cell 2010, 22, 1762–1776. [Google Scholar] [CrossRef] [PubMed]
  35. Rigo, G.; Ayaydin, F.; Tietz, O.; Zsigmond, L.; Kovacs, H.; Pay, A.; Salchert, K.; Darula, Z.; Medzihradszky, K.F.; Szabados, L.; et al. Inactivation of plasma membrane-localized CDPK-RELATED KINASE5 decelerates PIN2 exocytosis and root gravitropic response in Arabidopsis. Plant Cell 2013, 25, 1592–1608. [Google Scholar] [CrossRef] [PubMed]
  36. Chen, Q.; Liu, Y.; Maere, S.; Lee, E.; Van, I.G.; Xie, Z.; Xuan, W.; Lucas, J.; Vassileva, V.; Kitakura, S.; et al. A coherent transcriptional feed-forward motif model for mediating auxin-sensitive PIN3 expression during lateral root development. Nat. Commun. 2015, 6, 8821. [Google Scholar] [CrossRef] [PubMed]
  37. Willige, B.C.; Chory, J. A current perspective on the role of AGCVIII kinases in PIN-mediated apical hook development. Front. Plant Sci. 2015, 6, 767. [Google Scholar] [CrossRef]
  38. Rakusová, H.; Abbas, M.; Han, H.; Song, S.; Robert, H.S.; Friml, J. Termination of shoot gravitropic responses by auxin feedback on PIN3 polarity. Curr. Biol. 2016, 26, 3026–3032. [Google Scholar] [CrossRef] [PubMed]
  39. Friml, J.; Benková, E.; Blilou, I.; Wisniewska, J.; Hamann, T.; Ljung, K.; Woody, S.; Sandberg, G.; Scheres, B.; Jürgens, G.; et al. AtPIN4 mediates sink-driven auxin gradients and root patterning in Arabidopsis. Cell 2002, 108, 661–673. [Google Scholar] [CrossRef]
  40. Mravec, J.; Skůpa, P.; Bailly, A.; Hoyerová, K.; Krecek, P.; Bielach, A.; Petrášek, J.; Zhang, J.; Gaykova, V.; Stierhof, Y.D.; et al. Subcellular homeostasis of phytohormone auxin is mediated by the ER-localized PIN5 transporter. Nature 2009, 459, 1136–1140. [Google Scholar] [CrossRef]
  41. Simon, S.; Skůpa, P.; Viaene, T.; Zwiewka, M.; Tejos, R.; Klíma, P.; Čarná, M.; Rolčík, J.; Rycke, R.D.; Moreno, I.; et al. PIN6 auxin transporter at endoplasmic reticulum and plasma membrane mediates auxin homeostasis and organogenesis in Arabidopsis. New Phytol. 2016, 211, 65–74. [Google Scholar] [CrossRef] [PubMed]
  42. Cazzonelli, C.I.; Vanstraelen, M.; Simon, S.; Yin, K.; Carron-Arthur, A.; Nisar, N.; Tarle, G.; Cuttriss, A.J.; Searle, I.R.; Mathesius, J.; et al. Role of the Arabidopsis PIN6 auxin transporter in auxin homeostasis and auxin-mediated development. PLoS ONE 2013, 8, e70069. [Google Scholar] [CrossRef] [PubMed]
  43. Ditengou, F.A.; Gomes, D.; Nziengui, H.; Kochersperger, P.; Lasok, H.; Medeiros, V.; Paponov, I.V.; Nagy, S.K.; Nádai, T.V.; Mészáros, T.; et al. Characterization of auxin transporter PIN6 plasma membrane targeting reveals a function for PIN6 in plant bolting. New Phytol. 2017, 217, 1610–1624. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Rosquete, M.R.; Waidmann, S.; Kleine, V.J. PIN7 auxin carrier has a preferential role in terminating radial root expansion in Arabidopsis thaliana. Int. J. Mol. Sci. 2018, 19, 1238. [Google Scholar] [CrossRef] [PubMed]
  45. Zadnikova, P.; Petrasek, J.; Marhavy, P.; Raz, V.; Vandenbusche, F.; Ding, Z.; Schwarerova, K.; Morita, M.T.; Tasaka, M.; Van Der Straeten, D.; et al. Role of PIN-mediated auxin efflux in apical hook development of Arabidopsis thaliana. Development 2010, 137, 607–617. [Google Scholar] [CrossRef] [PubMed]
  46. Dal Bosco, C.; Dovzhenko, A.; Liu, X.; Woerner, N.; Rensch, T.; Eismann, M.; Eimer, S.; Hegermann, J.; Paponov, I.A.; Ruperti, B.; et al. The endoplasmic reticulum localized PIN8 is a pollen-specific auxin carrier involved in intracellular auxin homeostasis. Plant J. 2012, 71, 860–870. [Google Scholar] [CrossRef] [PubMed]
  47. Ding, Z.; Wang, B.; Moreno, I.; DupláKová, N.; Simon, S.; Carraro, N.; Reemmer, J.; Pěnčík, A.; Chen, X.; Tejos, R.; et al. ER-localized auxin transporter PIN8 regulates auxin homeostasis and male gametophyte development in Arabidopsis. Nat. Commun. 2012, 3, 941. [Google Scholar] [CrossRef] [PubMed]
  48. Carraro, N.; Tisdale-Orr, T.E.; Clouse, R.M.; Knoller, A.S.; Spicer, R. Diversification and expression of the PIN, AUX/LAX, and ABCB families of putative auxin transporters in Populus. Front. Plant Sci. 2012, 3, 17. [Google Scholar] [CrossRef]
  49. Chai, C.; Wang, Y.; Valliyodan, B.; Nguyen, H.T. Comprehensive analysis of the soybean (Glycine max) GmLAX auxin transporter gene family. Front. Plant Sci. 2016, 7, 282. [Google Scholar] [CrossRef]
  50. Shen, C.; Bai, Y.; Wang, S.; Zhang, S.; Wu, Y.; Chen, M.; Jiang, D.; Qi, Y. Expression profile of PIN, AUX/LAX and PGP auxin transporter gene families in Sorghum bicolor under phytohormone and abiotic stress. FEBS J. 2010, 277, 2954–2969. [Google Scholar] [CrossRef]
  51. Yue, R.; Tie, S.; Sun, T.; Zhang, L.; Yang, Y.; Qi, J.; Yan, S.; Han, X.; Wang, H.; Shen, C. Genome-wide identification and expression profiling analysis of ZmPIN, ZmPILS, ZmLAX and ZmABCB auxin transporter gene families in maize (Zea mays L.) under various abiotic stresses. PLoS ONE 2015, 10, e0118751. [Google Scholar] [CrossRef] [PubMed]
  52. Zhang, C.; Dong, W.; Huang, Z.A.; Cho, M.; Yu, Q.; Wu, C.; Yu, C. Genome-wide identification and expression analysis of the CaLAX and CaPIN gene families in pepper (Capsicum annuum L.) under various abiotic stresses and hormone treatments. Genome 2018, 61, 121–130. [Google Scholar] [CrossRef] [PubMed]
  53. He, P.; Zhao, P.; Wang, L.; Zhang, Y.; Wang, X.; Xiao, H.; Yu, J.; Xiao, G. The PIN gene family in cotton (Gossypium hirsutum): Genome-wide identification and gene expression analyses during root development and abiotic stress responses. BMC Genom. 2017. [Google Scholar] [CrossRef] [PubMed]
  54. Liu, C.; Zeng, L.; Zhu, S.; Wu, L.; Wang, Y.; Tang, S.; Wang, H.; Zheng, X.; Zhao, J.; Chen, X.; et al. Draft genome analysis provides insights into the fiber yield, crude protein biosynthesis, and vegetative growth of domesticated ramie (Boehmeria nivea L. Gaud). Dna Res. 2017, 25, 173–181. [Google Scholar] [CrossRef] [PubMed]
  55. Huala, E.; Dickerman, A.W.; Garcia-Hernandez, M.; Weems, D.; Reiser, L.; LaFord, F.; Hanley, D.; Kiphart, D.; Zhuang, M.; et al. The Arabidopsis Information Resource (TAIR): a comprehensive database and web-based information retrieval, analysis, and visualization system for a model plant. Nucleic Acids Res. 2001, 29, 102–105. [Google Scholar] [CrossRef]
  56. Li, T.; Qi, X.; Zeng, Q.; Xiang, Z.; He, N. MorusDB: A resource for mulberry genomics and genome biology. Database 2014, bau054. [Google Scholar] [CrossRef] [PubMed]
  57. Chen, J.; Pei, Z.; Dai, L.; Wang, B.; Liu, L.; An, X.; Peng, D. Transcriptome profiling using pyrosequencing shows genes associated with bast fiber development in ramie (Boehmeria nivea L.). BMC Genom. 2014, 15, 919. [Google Scholar] [CrossRef]
  58. Huang, X.; Chen, J.; Bao, Y.; Liu, L.; Jiang, H.; An, X.; Dai, L.; Wang, B.; Peng, D. Transcript Profiling Reveals Auxin and Cytokinin Signaling Pathways and Transcription Regulation during In Vitro Organogenesis of Ramie (Boehmeria nivea L. Gaud). PLoS ONE 2014, 9, e113768-24. [Google Scholar] [CrossRef]
  59. An, X.; Chen, J.; Zhang, J.; Liao, Y.; Dai, L.; Wang, B.; Liu, L.; Peng, D. Transcriptome Profiling and Identification of Transcription Factors in Ramie (Boehmeria nivea L. Gaud) in Response to PEG Treatment, Using Illumina Paired-End Sequencing Technology. Int. J. Mol. Sci. 2015, 16, 3493–3511. [Google Scholar] [CrossRef]
  60. Thompson, J.D.; Gibson, T.J.; Plewniak, F.; Jeanmougin, F.; Higgins, D.G. The Clustal_X Windows Interface: Flexible Strategies for Multiple Sequences Alignment Aided by Quality Analysis Tools. Nucleic Acids Res. 1997, 25, 4876–4882. [Google Scholar] [CrossRef]
  61. Open Reading Frame Finder. Available online: https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/orffinder/ (accessed on 6 August 2019).
  62. Benson, D.; Boguski, M.; Lipman, D.; Ostell, J.; Ouellette, B. GenBank. Nucleic Acids Res. 1998, 26, 1–7. [Google Scholar] [CrossRef] [PubMed]
  63. Luan, M.B.; Jian, J.B.; Chen, P.; Chen, J.H.; Chen, J.H.; Gao, Q.; Gao, G.; Zhou, J.H.; Chen, K.M.; Guang, X.M.; et al. Draft genome sequence of ramie, Boehmeria nivea (L.) Gaudich. Mol. Ecol. Resour. 2018, 18, 639–645. [Google Scholar] [CrossRef] [PubMed]
  64. Tamura, K.; Peterson, D.; Peterson, N.; Stecher, G.; Nei, M.; Kumar, S. MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol. Biol. Evol. 2011, 28, 2731–2739. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  65. Bailey, T.L.; Boden, M.; Buske, F.A.; Frith, M.; Grant, C.E.; Clementi, L.; Ren, J.; Li, W.W.; Noble, W.S. MEME SUITE: Tools for motif discovery and searching. Nucleic Acids Res. 2009, 37, W202–W208. [Google Scholar] [CrossRef] [PubMed]
  66. Krogh, A.; Larsson, B.; von Heijne, G.; Sonnhammer, E.L. Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. J. Mol. Biol. 2001, 305, 567–580. [Google Scholar] [CrossRef] [PubMed]
  67. Gasteiger, E.; Hoogland, C.; Gattiker, A.; Duvaud, S.; Wilkins, M.R.; Appel, R.D.; Bairoch, A. Protein identification and analysis tools on the ExPASy Server. Proteom. Protoc. Handb. 2005, 571–607. [Google Scholar] [CrossRef]
  68. Yu, C.; Chen, Y.; Lu, C.; Hwang, J. Prediction of protein subcellular localization. Proteins: Struct. Function Bioinformatics 2006, 64, 643–651. [Google Scholar] [CrossRef] [PubMed]
  69. Lescot, M.; Déhais, P.; Thijs, G.; Marchal, K.; Moreau, Y.; Peer, Y.V.D.; Rouzé, P.; Rombauts, S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002, 30, 325–327. [Google Scholar] [CrossRef] [PubMed]
  70. Chen, C.; Xia, R.; Chen, H.; He, Y. TBtools, a Toolkit for Biologists integrating various biological data handling tools with a user-friendly interface. BioRxiv 2018. [Google Scholar] [CrossRef]
  71. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
  72. Sparkes, I.A.; Runions, J.; Kearns, A.; Hawes, C. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat. Protoc. 2006, 1, 2019–2025. [Google Scholar] [CrossRef] [PubMed]
  73. Ibraheem, O.; Botha, C.E.J.; Bradley, G. In silico analysis of cis-acting regulatory elements in 5′ regulatory regions of sucrose transporter gene families in rice (Oryza sativa Japonica) and Arabidopsis thaliana. Comput. Biol. Chem. 2010, 34, 268–283. [Google Scholar] [CrossRef] [PubMed]
  74. Ganguly, A.; Park, M.; Kesawat, M.S.; Cho, H. Functional Analysis of the Hydrophilic Loop in Intracellular Trafficking of Arabidopsis PIN-FORMED Proteins. Plant Cell 2014, 26, 1570–1585. [Google Scholar] [CrossRef] [PubMed]
  75. Rakusová, H.; Gallego-Bartolomé, J.; Vanstraelen, M.; Robert, H.S.; Alabadí, D.; Blázquez, M.A.; Benková, E.; Friml, J. Polarization of PIN3-dependent auxin transport for hypocotyl gravitropic response in Arabidopsis thaliana. Plant J. 2011, 67, 817–826. [Google Scholar] [CrossRef] [PubMed]
  76. Sawchuk, M.G.; Edgar, A.; Scarpella, E. Patterning of Leaf Vein Networks by Convergent Auxin Transport Pathways. PLoS Genet. 2013, 9, e1003294-13. [Google Scholar] [CrossRef] [PubMed]
  77. Zhang, Y.; He, P.; Yang, Z.; Huang, G.; Wang, L.; Pang, C.; Xiao, H.; Zhao, P.; Yu, J.; Xiao, G. A Genome-Scale Analysis of the PIN Gene Family Reveals Its Functions in Cotton Fiber Development. Front. Plant Sci. 2017, 8. [Google Scholar] [CrossRef]
  78. Ghanashyam, C.; Jain, M. Role of auxin-responsive genes in biotic stress responses. Plant Signal. Behav. 2009, 4, 846–848. [Google Scholar] [CrossRef]
  79. Wang, Y.; Chai, C.; Valliyodan, B.; Maupin, C.; Annen, B.; Nguyen, H.T. Genome-wide analysis and expression profiling of the PIN auxin transporter gene family in soybean (Glycine max). BMC Genom. 2015, 16, 951. [Google Scholar] [CrossRef]
  80. Wang, S.; Shen, C.; Zhang, S.; Xu, Y.; Jiang, D.; Qi, Y. Analysis of subcellular localization of auxin carriers PIN, AUX/LAX and PGP in Sorghum bicolor. Plant Signal. Behav. 2014, 6, 2023–2025. [Google Scholar] [CrossRef]
  81. Xiong, L. Endogenous auxin and jasmonic acid levels are differentially modulated by abiotic stresses in rice. Front. Plant Sci. 2013, 4, 397. [Google Scholar] [CrossRef] [Green Version]
  82. Zhang, Q.; Li, J.; Zhang, W.; Yan, S.; Wang, R.; Zhao, J.; Li, Y.; Qi, Z.; Sun, Z.; Zhu, Z. The putative auxin efflux carrier OsPIN3t is involved in the drought stress response and drought tolerance. Plant J. 2012, 72, 805–816. [Google Scholar] [CrossRef] [PubMed]
  83. Peleg, Z.; Blumwald, E. Hormone balance and abiotic stress tolerance in crop plants. Curr. Opin. Plant Biol. 2011, 14, 290–295. [Google Scholar] [CrossRef] [PubMed]
  84. Liu, T.; Tang, Q.; Zhu, S. Analysis of climatic factors causing yield difference in ramie among different eco-regions of yangtze valley. J. Anhui Agric. Sci. 2011, 39. [Google Scholar] [CrossRef]
Figure 1. Phylogenetic relationship analysis of the (a) LAX and (b) PIN families among ramie, Arabidopsis, and mulberry. All branches are marked with bootstrap values. Different colored boxes indicate different subfamilies.
Figure 1. Phylogenetic relationship analysis of the (a) LAX and (b) PIN families among ramie, Arabidopsis, and mulberry. All branches are marked with bootstrap values. Different colored boxes indicate different subfamilies.
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Figure 2. Conserved domains of (a) AUX/LAX and (b) PIN proteins in ramie. The symbol heights indicate the relative frequency of each amino acid at that position.
Figure 2. Conserved domains of (a) AUX/LAX and (b) PIN proteins in ramie. The symbol heights indicate the relative frequency of each amino acid at that position.
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Figure 3. Transmembrane topology analysis of the (a) BnAUX/LAX and (b) BnPIN proteins in ramie. The predicted transmembrane helices are shown as red peaks.
Figure 3. Transmembrane topology analysis of the (a) BnAUX/LAX and (b) BnPIN proteins in ramie. The predicted transmembrane helices are shown as red peaks.
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Figure 4. The (a) gene structure and (b) cis-element prediction of BnAUX/LAX and BnPIN promoters. The exons are represented by yellow boxes, and the introns are represented by lines.
Figure 4. The (a) gene structure and (b) cis-element prediction of BnAUX/LAX and BnPIN promoters. The exons are represented by yellow boxes, and the introns are represented by lines.
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Figure 5. Expression pattern of (a) BnAUX/LAX and BnPIN genes in ramie tissues (leaf, shoot, root, bark) and (b) before and after IAA treatment (0 and 60 min). (* t-test, p-value < 0.1, ** p-value < 0.01, *** p-value < 0.001). The error bar represents the standard error.
Figure 5. Expression pattern of (a) BnAUX/LAX and BnPIN genes in ramie tissues (leaf, shoot, root, bark) and (b) before and after IAA treatment (0 and 60 min). (* t-test, p-value < 0.1, ** p-value < 0.01, *** p-value < 0.001). The error bar represents the standard error.
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Figure 6. Expression pattern of BnAUX/LAX and BnPIN genes in ramie by transcriptome analysis (blank spaces represent no data).
Figure 6. Expression pattern of BnAUX/LAX and BnPIN genes in ramie by transcriptome analysis (blank spaces represent no data).
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Figure 7. Subcellular localization of BnPIN5, BnAUX1, BnLAX1, and BnLAX2 proteins in tobacco leaf: (a) subcellular localization of the fused pEGAD-35S::BnPIN (BnAUX/LAX):GFP in tobacco leaf cells. The pEGAD-35S::GFP construct was used as the control. (b) The fusion proteins were transiently expressed in tobacco epidermis cells. Merged overlays of bright-field and green fluorescence images are shown. The scale bars are 20 μm.
Figure 7. Subcellular localization of BnPIN5, BnAUX1, BnLAX1, and BnLAX2 proteins in tobacco leaf: (a) subcellular localization of the fused pEGAD-35S::BnPIN (BnAUX/LAX):GFP in tobacco leaf cells. The pEGAD-35S::GFP construct was used as the control. (b) The fusion proteins were transiently expressed in tobacco epidermis cells. Merged overlays of bright-field and green fluorescence images are shown. The scale bars are 20 μm.
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Table 1. Auxin transport-related genes in ramie and their CELLO localizations.
Table 1. Auxin transport-related genes in ramie and their CELLO localizations.
GeneGenBank NumberCDS (bp)Predicted Protein Length (aa)Molecular WeightTheoretical pICELLO Localization
BnAUX1KR139986147048954960.838.41PlasmaMembrane (4.989)
BnLAX1KR139987146748854610.798.77PlasmaMembrane (4.989)
BnLAX2KR139988149149655944.479.01PlasmaMembrane (4.970)
BnLAX3KR139989141046953098.199.18PlasmaMembrane (4.970)
BnPIN1aKR139990177659163313.038.70PlasmaMembrane (4.225)
BnPIN1bKR139991186962267970.128.64PlasmaMembrane (4.601)
BnPIN3KR139992202267372588.557.30PlasmaMembrane (3.607)
BnPIN5KR139993108636139707.956.30PlasmaMembrane (3.607)
BnPIN6KR139994165655160102.288.96PlasmaMembrane (3.607)
BnPIN8KR139995108035939279.199.40PlasmaMembrane (3.607)

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Bao, Y.; Huang, X.; Rehman, M.; Wang, Y.; Wang, B.; Peng, D. Identification and Expression Analysis of the PIN and AUX/LAX Gene Families in Ramie (Boehmeria nivea L. Gaud). Agronomy 2019, 9, 435. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy9080435

AMA Style

Bao Y, Huang X, Rehman M, Wang Y, Wang B, Peng D. Identification and Expression Analysis of the PIN and AUX/LAX Gene Families in Ramie (Boehmeria nivea L. Gaud). Agronomy. 2019; 9(8):435. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy9080435

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Bao, Yaning, Xing Huang, Muzammal Rehman, Yunhe Wang, Bo Wang, and Dingxiang Peng. 2019. "Identification and Expression Analysis of the PIN and AUX/LAX Gene Families in Ramie (Boehmeria nivea L. Gaud)" Agronomy 9, no. 8: 435. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy9080435

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