Abstract
Auxin polar transport is crucial in regulating plant growth and patterning. As auxin efflux carriers, the PIN FORMED (PIN) proteins are responsible for transportation of auxin out of the cell. There are eight and ten PIN members in Arabidopsis (AtPIN) and Medicago truncatula (MtPIN), respectively. Compared with MtPIN10/SMOOTH LEAF MARGIN1 (SLM1), MtPIN4 exhibits a closer relationship with AtPIN1 based phylogenetic analysis. In addition, the gene structure and distribution of transmembrane segments of MtPIN4, MtPIN5 and MtPIN10/SLM1 are similar, implying possible redundant roles among them. However, analysis using Gene Expression Atlas revealed different expression patterns among MtPIN4, MtPIN5 and MtPIN10/SLM1. Loss of function of MtPIN10/SLM1 in M. truncatula resulted in pleiotropic phenotypes in different organs, which are similar with the defects in the pin1 mutant of Arabidopsis, suggesting that the MtPIN10/SLM1 is a putative ortholog of AtPIN1. MtPIN4, MtPIN5 and MtPIN10/SLM1 may have limited redundant functions in the development of M. truncatula. The creation of double and triple mutants will help to elucidate their potential roles in auxin transport and plant development.
Keywords: Tnt1-tagged mutant, Arabidopsis, auxin, PIN-FORMED
The phytohormone auxin plays a key role in regulating plant growth and development. Although the rate of auxin biosynthesis is important for the overall auxin status of the plant, it is the fine concentration gradients across cells that have powerful effects on plant development.1 The transportation of auxin to specific tissues triggers a signaling cascade and causes various developmental responses.2 Auxin distribution is dependent on its polar transport which is mediated by influx carriers and auxin efflux carriers. AUXIN RESISTANT 1 (AUX1) and LIKE-AUX1 (LAX) proteins are the auxin import carriers that allow protonated auxin to enter the cell.3-5 The PIN FORMED (PIN) proteins belong to a family of polarly localized plasma membrane proteins and act as auxin efflux carriers. Most of the members in the PIN family are able to transport auxin out of the cell.2,6 The Arabidopsis thaliana PIN family consists of eight members (AtPIN1 to AtPIN8). They are expressed specifically in auxin-transporting tissues and cells and asymmetrically localized in the plasma membranes of these cells.2 This polar subcellular localization is necessary for the generation of auxin activity gradients that are important for plant development, especially for organ initiation.7,8 In the PIN family of Arabidopsis, the first member that was characterized is AtPIN1.9 AtPIN1 is localized at the L1 surface cell layer of shoot apical meristem (SAM), directing auxin flow toward the summit of the meristem. The auxin convergence point at the periphery of the SAM marks the sites of initiating lateral organ primordia.10,11 Loss of function of AtPIN1 in Arabidopsis results in the lack of almost all flowers or lateral organs along the inflorescence and shows defects in the separation of lateral organs.8,12
Medicago truncatula is a compound leaf species and serves as a model organism for legumes. Although ten auxin efflux carriers (MtPIN1 to MtPIN10) were isolated from M. truncatula,13 only MtPIN10/SMOOTH LEAF MARGIN 1 (SLM1) has been functionally characterized.14 The PIN10/SLM mutant was identified by screening the Tnt1 retrotransposon-tagged M. truncatula population. Phylogenetic analyses of the PIN family members from Arabidopsis and M. truncatula revealed that MtPIN4, MtPIN5 and MtPIN10 are in a clade with AtPIN1, implying a close evolutionary relationship among them (Fig. 1A). Based on sequence alignments of predicted amino acids, MtPIN4 and MtPIN5 showed 72% identity, while MtPIN10 had 66% and 64% identities with MtPIN4 and MtPIN5, respectively. AtPIN1 had 71%, 65% and 65% amino acid identity with MtPIN4, MtPIN5 and MtPIN10/SLM1, respectively. Therefore, MtPIN4 appears to be more closely related to AtPIN1 compared with MtPIN10/SLM1. Intron/exon analysis displayed an overall deduced gene structure with a shift of intro/exon boundaries in AtPIN1, MtPIN4, MtPIN5 and MtPIN10/SLM1 (Fig. 1B). MtPIN5 has the shortest coding region (Fig. 1B). Transmembrane prediction analysis showed that all the four proteins are comprised of conserved N-terminal and C-terminal regions of transmembrance segments, and a variable middle region. However, different characteristics, including the number of transmembrance domains and the localization of middle loop, are also observed (Fig. 1C).
Figure 1.
Bioinformatic analyses of AtPIN1, MtPIN4, MtPIN5 and MtPIN10/SLM1. (A) Phylogenetic analysis of PIN in Arabidopsis and M. truncatula. Alignments were performed using ClustalW with default parameters. The phylogenetic tree was constructed by the MEGA2 program with 1,000 bootstrap replicates. (B) Deduced intron/exon structure of PIN genes. Boxes represent exons and lines represent introns. (C) The predication of transmembrane domains in PIN using TMHMM: http://www.cbs.dtu.dk/services/TMHMM-2.0. (D) Expression profiling of the MtPIN4, MtPIN5 and MtPIN10/SLM1 transcript (mtgea.noble.org/v2).
The expression of AtPIN1 is detected in seedlings, leaves, stems, flowers and roots in Arabidopsis.9 To compare the transcript levels of MtPIN4, MtPIN5 and MtPIN10/SLM1 in different organs of M. truncatula, their expression patterns were analyzed using the M. truncatula Gene Expression Atlas (Fig. 1D). The probe sets Mtr.1076.1.S1_at, Mtr.17296.1.S1_at and Mtr.47942.1.S1_at represent MtPIN4, MtPIN5 and MtPIN10/SLM1 in the microarray chip, respectively. The analysis revealed that the overall expression level of MtPIN4 is much higher than that of MtPIN10/SLM1 and their expression patterns showed a similar trend. Furthermore, the expression level of MtPIN5 is very low in most organs except in developing seeds and seed coat. Previous studies proposed that MtPIN5 is probably a duplicated but silenced gene of MtPIN4 during evolution.13 However, the obvious increase of transcript level of MtPIN5 but not MtPIN4 and MtPIN10/SLM1 in seed coat may imply a special role of MtPIN5 in this organ.
Loss of function of MtPIN10/SLM1 leads to obvious defects in the initiation and separation of lateral organs in M. truncatula.14 Such defects are similar to the classical Arabidopsis pin1 mutant phenotype.8,9,12,14 The phenotypic data and the mutant complementation analysis suggest that MtPIN10/SLM1 is the putative ortholog of AtPIN1. However, MtPIN4 seems to have a closer relationship with AtPIN1 based on phylogenic analysis. Therefore, we propose that MtPIN4, MtPIN5, and MtPIN10/SLM1 have limited redundant roles. It will be interesting to identify the mutants of MtPIN4 and MtPIN5, and to investigate their potential redundant functions in auxin transport and plant development by creating double and triple mutants. Furthermore, promoter analysis of these three MtPIN genes will help to uncover their expression patterns and developmental roles in M. truncatula.
Acknowledgments
The work described in this addendum was supported by the Samuel Roberts Noble Foundation.
Footnotes
Previously published online: www.landesbioscience.com/journals/psb/article/17508
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