Genome-wide analysis and expression profiling of the PIN auxin transporter gene family in soybean (Glycine max)
- Yongqin Wang†1,
- Chenglin Chai†1,
- Babu Valliyodan1,
- Christine Maupin1,
- Brad Annen1 and
- Henry T. Nguyen1Email author
© Wang et al. 2015
Received: 16 July 2015
Accepted: 26 October 2015
Published: 16 November 2015
The plant phytohormone auxin controls many aspects of plant growth and development, which largely depends on its uneven distribution in plant tissues. Transmembrane proteins of the PIN family are auxin efflux facilitators. They play a key role in polar auxin transport and are associated with auxin asymmetrical distribution in plants. PIN genes have been characterized in several plant species, while comprehensive analysis of this gene family in soybean has not been reported yet.
In this study, twenty-three members of the PIN gene family were identified in the soybean genome through homology searches. Analysis of chromosome distribution and phylogenetic relationships of the soybean PIN genes indicated nine pairs of duplicated genes and a legume specific subfamily. Organ/tissue expression patterns and promoter activity assays of the soybean PINs suggested redundant functions for most duplicated genes and complementary and tissue-specific functions during development for non-duplicated genes. The soybean PIN genes were differentially regulated by various abiotic stresses and phytohormone stimuli, implying crosstalk between auxin and abiotic stress signaling pathways. This was further supported by the altered auxin distribution under these conditions as revealed by DR5::GUS transgenic soybean hairy root. Our data indicates that GmPIN9, a legume-specific PIN gene, which was responsive to several abiotic stresses, might play a role in auxin re-distribution in soybean root under abiotic stress conditions.
This study provided the first comprehensive analysis of the soybean PIN gene family. Information on phylogenetic relationships, gene structure, protein profiles and expression profiles of the soybean PIN genes in different tissues and under various abiotic stress treatments helps to identity candidates with potential roles in specific developmental processes and/or environmental stress conditions. Our study advances our understanding of plant responses to abiotic stresses and serves as a basis for uncovering the biological role of PIN genes in soybean development and adaption to adverse environments.
Plant phytohormones are small signaling molecules that are synthesized within plant and control many aspects of plant growth and development, as well as plant responses to environmental cues. Auxin is the most studied and the most important plant hormone. It plays crucial roles in apical meristem maintenance, axillary meristem formation, growth, as well as phototropism, gravitropism and hydrotropism. Executing the multiple roles of auxin is largely dependent on its uneven distribution in plant, which is achieved through an active process called polar auxin transport mediated by plasma membrane auxin transporter proteins [1, 2]. Three major gene families of auxin transporters have been found in plants. Of them the plant specific PIN-FORMED (PIN) auxin efflux facilitators are key players in this process, which work together with the AUXIN1 (AUX1)/LIKE AUX1 (LAX) influx carriers and the phosphoglycoprotein (PGP/MDR⁄ABCB) efflux/influx transporters [3, 4]. The asymmetric subcellular localization of PIN proteins determines the directionality of intercellular auxin flow and the differential distribution of auxin within plant tissue, thereby controlling various plant developmental processes [3, 5, 6].
Our current knowledge about PIN-dependent polar auxin transport in plant mostly comes from the extensive investigation of the PIN gene family in Arabidopsis (Arabidopsis thaliana), which includes 8 members . Five of the Arabidopsis PINs (PIN1-4 and PIN7) are located in the plasma membrane and they play a prominent role in the directional, cell-to-cell auxin transport . Their spatiotemporal expression patterns and the auxin-dependent cross regulation of their expressions make them functionally redundant and complementary in a variety of plant developmental processes, including embryogenesis, organogenesis, tissue differentiation and tropism . On the contrary, the endoplasmic reticulum (ER)-localized PIN5, 6, and 8 are not directly involved in the cell-to-cell auxin transport, but play a role in intracellular regulation of auxin homeostasis by working together with members of the PIN-LIKE auxin efflux carriers [9–13]. Besides their obvious role in many developmental processes, results from Arabidopsis show that PIN proteins are also involved in the crosstalk of auxin, ethylene, cytokinin and strigolactone in root development, where PIN proteins serve as a playground for the integration of hormone signaling through regulation of intracellular PIN protein trafficking and then subcellular polar localization . Recently, the PIN gene family has been characterized from rice, sorghum, maize, and potato [15–19]. Transcriptional profiling analyses in rice, sorghum and maize suggested that some PIN genes from these plant species might mediate the crosstalk between auxin, other hormones and abiotic stresses [15–17].
Soybean is one of the most widely grown crops in the world. It is the most important source of vegetable protein and oil for humans, the most preferable protein source for farm animals, and currently the major feedstock for biodiesel production. With the rapid growth of global population and environmental degradation, improving soybean yield is a crucial task to meet the human demand for food and energy. Considering the importance of PIN genes in plant growth regulation and in plant response to abiotic stress environments, we carried out genome-wide comprehensive analysis of the soybean PIN auxin efflux transporter gene family. Their tissue expression patterns and expression profiling under hormonal treatments such as auxin and abscisic acid (ABA), and abiotic stresses including drought, salt and dehydration were analyzed. Our research identified the soybean PINs associated with abiotic stress responses. Some of them might be ideal candidates for further investigation.
Results and Discussion
Identification and phylogenetic analysis of the soybean GmPINs
Twenty-four putative GmPIN loci have been found through BLAST searches of the Glycine max reference genome (v1.1) by using A. thaliana PIN protein sequences, including two truncated loci, Glyma13g09026 and Glyma13g09043, which were located in the same region of the chromosome. They were treated as a single locus (Glyma13g09030) in the annotation of Glycine max version 1.0. We adopted the gene model of Glyma13g09030 in our following analysis because Glyma13g09026 (encoding a 126-amino acid peptide) and Glyma13g09043 (encoding a 350-amino acid peptide) show high sequence similarities with the C terminal and N terminal of Glyma14g27900, respectively. These two loci might be evolved from a single locus, and further experiments are needed to verify this gene model. Therefore, a total of 23 members of the soybean PIN family have been identified. Using the same approach, 16, 12 and 5 putative PIN members were identified from common bean (Phaseolus vulgaris), Medicago truncatula and Lotus japonicus, respectively. Besides the previous characterized MtPIN1-7 , five new full-length sequences were identified in the Medicago truncatula v4 release.
Number of PIN genes in eight plant species
Chromosomal distribution, gene structure and protein profiles of GmPINs
Tissue-specific expression profile of GmPINs
As shown in Fig. 5, a different combination of GmPIN expression was observed in each tissue types, such as members of GmPIN1, GmPIN3 and GmPIN6 in shoot apical meristem, and the homologous genes of PIN1 and PIN2 in root tip. This suggests that these GmPINs work cooperatively. The dynamic temporal and spatial expression of these GmPINs may be critical for the developmental process of each tissue. Though expressed in the same tissue and at the same time, the cell type-specific expression pattern, subcellular polarity and subcellular localization of those genes can be different, which has been elegantly evidenced by experiments in Arabidopsis [5, 7]. The similar expression patterns of duplicated genes or genes from the same PIN group indicate functional redundancy, while synergistic expression of GmPINs from different group suggests functional complementation. Both might contribute to the flexibility and variation during soybean evolution. Functional redundancy could reduce the selection pressure on duplicated genes, which might lead to novel function, loss of function, or loss of expression (such as GmPIN8c, GmPIN8d, GmPIN9a, GmPIN9b, and GmPIN9c).
Expression of GmPINs in response to drought, salt and dehydration
Current study indicates that the PIN auxin efflux transporters were evolved as key players in a plant's adaptations to their growing environment by responding to environmental and endogenous signals at both transcriptional and post-transcriptional levels . Drought is one of the prime abiotic stresses worldwide, and is also the major constraint to soybean production, accounting for 40 % of yield loss . Salinity is another significant stressor causing serious yield loss in salt-affected area, occupying 20 % of irrigated land in the world .
Our data demonstrated most GmPINs were responsive to certain water deficit conditions at the transcriptional level, generally in a tissue-specific, time- and stress magnitude-sensitive mode, suggesting that soybean responds to water deficit stress through a very complex regulation network, which necessitates coordinated regulation of most GmPINs. As in soybean, many PIN genes in sorghum and maize were found to be transcriptionally responsive to various abiotic stresses, including salt and drought [15, 17]. PIN genes might be commonly used for plants from different species to adapt to various abiotic stress conditions. Although a huge body of evidence has demonstrated the importance of PIN proteins in many developmental processes and in response to environmental signals such as light and gravity [1, 2, 4, 8], there is limited information on their role in abiotic stresses and the underlying molecular mechanisms. Recently, it was reported that PIN2 in Arabidopsis was required to maintain root growth under alkaline stress conditions by modulate proton [H+] secretion . Another study indicated that the affected intracellular trafficking of PIN2 and PIN3 proteins in Arabidopsis might be responsible for the inhibited auxin polar transport under cold stress conditions .
Expression of GmPINs in response to ABA and auxin
Exposure of plants to abiotic stress conditions elicits ABA accumulation, which then triggers a series of physiological, biological and molecular changes for plants to adapt to adverse environments. Evidence from Arabidopsis and rice supported that ABA accumulation modulated auxin transport in the root tip, which was critical for maintaining root growth under water stress condition . Besides ABA and auxin, many other hormones are involved in modulating plant’s response and adaption to environmental stresses [36, 37], as well as in controlling PIN gene action in many developmental processes [6, 16, 17, 26]. Regulation of PINs at the transcriptional and/or posttranscriptional level, including spatial and temporal expression pattern, subcellular polar localization, intracellular trafficking and recycling, and degradation, has been employed by plants to control many growth and developmental processes [5–8, 26]. Plants may use the same or similar mechanisms to adapt to stress conditions.
GmPIN promoter activity in transgenic soybean hairy root
In soybean roots, the overall similar patterns in promoter activity of the duplicated genes, such as GmPIN1b-1c, GmPIN2a-2b, and GmPIN3a-3b, further suggest their function redundancy. Members from PIN1-3 groups and GmPIN9d may play very important roles in soybean root development due to their relatively strong expression in this tissue. Notably, the patterns of promoter activity in soybean roots for the detected GmPINs were different from those of their orthologous genes in Arabidopsis and rice [8, 16]. The large gene number, various tissue-specific expression patterns and versatile regulatory modes under internal and external cues make it very complicated to explore the specific role of a certain GmPIN gene in soybean development. Further cellular and subcellular localization of their proteins in plant and gene-specific or a group of duplicated gene-specific knockout transgenic analysis may be helpful to unravel their functions.
Auxin distribution and GmPIN9d promoter activity in soybean root in response to environmental signals
Changes in DR5 promoter activity reflected altered auxin distribution or signaling. DR5 and GmPIN9 showed overall similar patterns in changes of promoter activity under PEG and salt treatments. This strongly indicates that GmPIN9 might play a role in auxin re-distribution under these conditions, probably working together with other GmPINs and auxin transporters from other gene families. Besides auxin transport, some auxin signaling components might also be involved in these responses. For example, some members of the auxin response factor transcription factor family in soybean were found transcriptionally regulated under water-deficit conditions .
In this study, the soybean PIN auxin transporter gene family was comprehensively analyzed, including phylogeny, chromosomal distribution, gene structure, protein profiles, expression profiles in various tissues and under various abiotic stress conditions and hormone treatments, and promoter activity assay in transgenic soybean root. Eighteen out of the 23 members in the soybean PIN gene family exist as duplicated gene pairs originating from the glycine-specific whole-genome duplication event. High potential functional redundancy of duplicated genes or genes from the same PIN group was implied from high similarities of encoded amino acids, gene structure, tissue-specific expression pattern, and promoter activity in soybean root. However, the versatile differential expression modes under abiotic stress conditions indicated specific gene function at certain environmental conditions. The soybean PIN genes were responsive to a complex network of internal and external signals, and thereby controlled the auxin re-distribution, which finally will lead to the adjustment of growth and developmental processes to adapt to the ever-changing environments. Further in-depth functional analysis of the biological roles of GmPIN genes will enhance our understanding of plant response to abiotic stresses and aid in development of stress resistant crops.
Identification of PIN auxin efflux carriers from soybean and other legumes
Putative soybean and common bean PIN auxin efflux carriers were identified by BLAST searches against the corresponding reference genome at Phytozome (v9.1)  using A. thaliana PIN protein sequences as queries. Following this approach, putative PIN members were identified from the Medicago truncatula genome (v4) , and the Lotus japonicus genome assembly build 2.5 . Protein sequences were downloaded for all identified putative PINs. See Additional file 3: Table S2 for accession numbers of all sequences used in this study.
Phylogenetic analysis and chromosomal mapping
Sequence alignments of all identified PINs from four legume species in this study and PINs with published data from Arabidopsis, rice, maize and sorghum were performed using the online software Clustal Omega . Result of the sequence alignments was then used to construct the unrooted phylogenetic tree by the neighbor-joining method with a bootstrap analysis of 1000 replicates using MEGA 5.2 . Chromosomal position information of GmPINs was obtained from gene annotation (v1.1), and the relative localization of each GmPIN was drawn on their respective chromosomes from the top to the bottom.
Gene structure and protein profile analysis
Gene exon-intron structure information of GmPINs was retrieved from Phytozome v9.1, and gene structure schematic diagram was drawn by using the Gene Structure Display Server . Protein transmembrane topology was predicted by using TMHHM Server v2.0 . Protein length, molecular weight and isoelectric point of GmPINs were analyzed by the Lasergene v7.1 software. Protein subcellular localization was predicted by WoLF PSORT .
Plant growth, stress and hormonal treatments and tissue collection
The soybean cultivar, Williams 82, was used in this study for the expression profiling analysis, promoter cloning and hairy root transformation. Plants were grown under the same greenhouse conditions as reported . For tissue/organ-specific expression profiling analysis, roots, mature leaves, immature leaves and stems were collected from V1 stage seedlings, and flowers, young pods (0.5 to 2 cm in length), and seeds at 14 and 21 days after flowering were collected from R3 to R6 growth stages. The same methods were followed for drought, dehydration, salt (250 mM) and ABA (150 μM) treatments . IAA treatments were conducted using the same method as ABA treatments except 50 μM IAA was used instead of ABA. Shoots and roots were collected separately and tissues from three plants were pooled as one sample after drought treatments. Whole plants were collected at different time points after dehydration and salt treatments. For hormone treatments, shoots and roots from single plants were collected separately at 0.5 hour (h), 1 h, 3 h and 5 h after treatments. Samples were frozen immediately in liquid nitrogen after collection and kept at −80 °C until use.
Total RNA extraction, design of GmPIN gene-specific primers for quantitative PCR (Additional file 4: Table S3), and qRT-PCR analysis were conducted following the standard methods . All qPCR analyses have three biological replicates and two technical replicates.
Promoter cloning and vector construction
Promoters (1,938 to 3,439 bp upstream of the start codon) of 10 GmPINs were amplified by PCR using Phusion high-fidelity DNA polymerase (Thermo Scientific, USA), gene-specific primers (Additional file 5: Table S4), and soybean genomic DNA extracted from two-week old seedlings using the CTAB method . PCR products were cloned into the Gateway pDONR™/Zeo vector (Invitrogen, USA), sequenced and then recombined into the destination vector pMDC163  to produce promoter::GUS expression cassettes via LR reactions (Invitrogen, USA). The DR5 synthetic promoter  was also constructed into pMDC163. All plant expression vectors were transformed into Agrobacterium rhizogenes K599 by electroporation.
Soybean hairy root transformation and GUS staining
Soybean hairy root transformation was performed according to references with some modifications [52, 53]. Half of the Murashige & Skoog (MS) medium, with the addition of hygromycin (25 mg/L) as a selective agent for transgenic roots, was used in the hairy root subculture. For PEG and salt treatments, 250 g/L PEG 8,000 and 75 mM NaCl was added in the media. For treatments with auxin and ABA, the concentration of IAA and ABA was 10−7 M. Lateral roots of similar size from the same transgenic root were used for all the treatments and control. The treatments lasted for 24 hours. At least 5 independent transgenic roots were used as biological replicates.
GUS staining was performed according to the standard protocols . The staining time was 30 minutes and 4 hours for DR5::GUS and GmPIN promoter::GUS transgenic roots, respectively. Root images were developed using a Leica S6 D stereomicroscope (Leica Microsystems, Switzerland) and Leica EC3 digital camera (Leica Microsystems, Switzerland).
We thank Dr. Thomas J. Guilfoyle (University of Missouri) for sharing the DR5 promoter, Dr. Xiaoli Guo (University of Missouri) for help with soybean hairy root transformation, and Theresa Musket (University of Missouri) for carefully editing this manuscript. This research was funded by the Missouri Soybean Merchandising Council Grant number 275 F (Translational Genomics for Drought Tolerance in Soybean).
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