ATP-binding Cassette Subfamily B Research Articles

Genome-wide identification of ATP-binding cassette transporter B subfamily, focusing on its structure, evolution and rearrangement in ciliates.

ATP-binding cassette subfamily B (ABCB) has been implicated in various essential functions such as multidrug resistance, auxin transport and heavy metal tolerance in animals and plants. However, the functions, the genomic distribution and the evolutionary history have not been characterized systematically in lower eukaryotes. As a lineage of highly specialized unicellular eukaryotes, ciliates have extremely diverse genomic features including nuclear dimorphism. To further understand the genomic structure and evolutionary history of this gene family, we investigated the ABCB gene subfamily in 11 ciliates. The results demonstrate that there is evidence of substantial gene duplication, which has occurred by different mechanisms in different species. These gene duplicates show consistent purifying selection, suggesting functional constraint, in all but one species, where positive selection may be acting to generate novel function. We also compare the gene structures in the micronuclear and macronuclear genomes and find no gene scrambling during genome rearrangement, despite the abundance of such scrambling in two of our focal species. These results lay the foundation for future analyses of the function of these genes and the mechanisms responsible for their evolution across diverse eukaryotic lineages.

Genome-Wide Identification, Expansion, Evolution, and Expression Analysis Reveals ABCB Genes Important for Secondary Cell Wall Development in Moso Bamboo (Phyllostachys edulis)

The ATP-binding cassette subfamily B (ABCB) is an important transporter family, and many members are well known for their auxin transport function. However, reports on the function of the ABCB genes during Moso bamboo development are few. In this study, we identified and characterized 37 PhABCB genes in Moso bamboo and classified them into five subgroups, Ⅰ–Ⅴ. We further observed gene family expansion and gene loss events during the evolution of the PhABCB gene family. It was found that the expansion of the PhABCB family was mainly attributed to the whole-genome duplication and DNA-transposed duplication models. Gene co-expression analysis and quantitative real-time PCR revealed that several PhABCB genes may be involved in the development of the secondary cell wall (SCW) during the rapid growth of Moso bamboo. Through examining their expression in different parts of the bamboo internode where the cell walls exhibited different developmental stages, the roles of eight candidate PhABCBs in the SCW development were further characterized. Of the eight PhABCB genes, PhABCB7, PhABCB11, PhABCB14, and PhABCB21 may be involved in the SCW biogenesis in Moso bamboo. This study provides the basis for discovering the potential role of PhABCB genes in Moso bamboo cell wall development; further studies are needed to elucidate how these PhABCBs function in SCW development by regulating the polar transport of auxin.

Functional analysis revealed the involvement of ZmABCB15 in resistance to rice black-streaked dwarf virus infection

BackgroundMaize rough dwarf disease (MRDD), caused by rice black-streaked dwarf virus (RBSDV) belonging to the Fijivirus genus, seriously threatens maize production worldwide. Three susceptible varieties (Ye478, Zheng 58, and Zhengdan 958) and two resistant varieties (P138 and Chang7–2) were used in our study.ResultsA set of ATP-binding cassette subfamily B (ABCB) transporter genes were screened to evaluate their possible involvements in RBSDV resistance. In the present study, ZmABCB15, an ABCB transporter family member, was cloned and functionally identified. Expression analysis showed that ZmABCB15 was significantly induced in the resistant varieties, not in the susceptible varieties, suggesting its involvement in resistance to the RBSDV infection. ZmABCB15 gene encodes a putative polar auxin transporter containing two trans-membrane domains and two P-loop nucleotide-binding domains. Transient expression analysis indicated that ZmABCB15 is a cell membrance localized protein. Over-expression of ZmABCB15 enhanced the resistance by repressing the RBSDV replication ratio. ZmABCB15 might participate in the RBSDV resistance by affecting the homeostasis of active and inactive auxins in RBSDV infected seedlings.ConclusionsPolar auxin transport might participate in the RBSDV resistance by affecting the distribution of endogenous auxin among tissues. Our data showed the involvement of polar auxin transport in RBSDV resistance and provided novel mechanism underlying the auxin-mediated disease control technology.

Systems approaches reveal that ABCB and PIN proteins mediate co-dependent auxin efflux.

Members of the B family of membrane-bound ATP-binding cassette (ABC) transporters represent key components of the auxin efflux machinery in plants. Over the last two decades, experimental studies have shown that modifying ATP-binding cassette sub-family B (ABCB) expression affects auxin distribution and plant phenotypes. However, precisely how ABCB proteins transport auxin in conjunction with the more widely studied family of PIN-formed (PIN) auxin efflux transporters is unclear, and studies using heterologous systems have produced conflicting results. Here, we integrate ABCB localization data into a multicellular model of auxin transport in the Arabidopsis thaliana root tip to predict how ABCB-mediated auxin transport impacts organ-scale auxin distribution. We use our model to test five potential ABCB–PIN regulatory interactions, simulating the auxin dynamics for each interaction and quantitatively comparing the predictions with experimental images of the DII-VENUS auxin reporter in wild-type and abcb single and double loss-of-function mutants. Only specific ABCB–PIN regulatory interactions result in predictions that recreate the experimentally observed DII-VENUS distributions and long-distance auxin transport. Our results suggest that ABCBs enable auxin efflux independently of PINs; however, PIN-mediated auxin efflux is predominantly through a co-dependent efflux where co-localized with ABCBs.

Genome-wide identification and expression profiles of ABCB gene family in Chinese hickory (Carya cathayensis Sarg.) during grafting

Chinese hickory (Carya cathayensis Sarg.) is an important nut tree species native to China. Excessive plant height and long juvenile phase has restricted development of its industry. Recently, grafting has been used increasingly in production practice of this species to solve the problems above. Previous studies have proved the importance of auxin during Chinese hickory grafting. However, the function of ATP-binding cassette subfamily B (ABCB) genes during Chinese hickory grafting is less studied. In this study, 23 ABCB genes were identified and characterized in Chinese hickory (CcABCBs). The expression profiles of auxin-related ABCBs among tissues, under auxin-related phytohormone treatments and during grafting were determined. CcABCB proteins were divided into half-size and full-size transporters. Many phytohormone-related cis-acting regulatory elements were detected on the promoters of CcABCB genes. Four CcABCB genes homologous to auxin-related AtABCB1, 6, 19 and 20 in Arabidopsis were selected for expression analysis. The four genes displayed varying expression patterns in different tissues of Chinese hickory. Expressions of the four CcABCB genes were regulated by auxin-related phytohormones to varying degrees. Expression levels of the four genes were significantly changed at different stages of grafting, especially 7 days after grafting, indicating their involvement of auxin homeostasis regulation during grafting. In addition, the expressions of CcABCB1 were regulated by IAA and NPA treatments during grafting in comparison with CK treatment, while expressions of the other 3 CcABCB genes were slightly affected. This study will lay the foundation for understanding the potential regulatory roles of CcABCB genes during Chinese hickory grafting.

ABCB transporters in a leaf beetle respond to sequestered plant toxins.

Phytophagous insects can tolerate and detoxify toxic compounds present in their host plants and have evolved intricate adaptations to this end. Some insects even sequester the toxins for their defence. This necessitates specific mechanisms, especially carrier proteins that regulate uptake and transport to specific storage sites or protect sensitive tissues from noxious compounds. We identified three ATP-binding cassette subfamily B (ABCB) transporters from the transcriptome of the cardenolide-sequestering leaf beetle Chrysochus auratus and analysed their functional role in the sequestration process. These were heterologously expressed and tested for their ability to interact with various potential substrates: verapamil (standard ABCB substrate), the cardenolides digoxin (commonly used), cymarin (present in the species's host plant) and calotropin (present in the ancestral host plants). Verapamil stimulated all three ABCBs and each was activated by at least one cardenolide, however, they differed as to which they were activated by. While the expression of the most versatile transporter fits with a protective role in the blood-brain barrier, the one specific for cymarin shows an extreme abundance in the elytra, coinciding with the location of the defensive glands. Our data thus suggest a key role of ABCBs in the transport network needed for cardenolide sequestration.

Investigation of physiological and molecular mechanisms conferring diurnal variation in auxinic herbicide efficacy.

The efficacy of auxinic herbicides, a valuable weed control tool for growers worldwide, has been shown to vary with the time of day in which applications are made. However, little is known about the mechanisms causing this phenomenon. Investigating the differential in planta behavior of these herbicides across different times of application may grant an ability to advise which properties of auxinic herbicides are desirable when applications must be made around the clock. Radiolabeled herbicide experiments demonstrated a likely increase in ATP-binding cassette subfamily B (ABCB)-mediated 2,4-D and dicamba transport in Palmer amaranth (Amaranthus palmeri S. Watson) at simulated dawn compared to mid-day, as dose response models indicated that many orders of magnitude higher concentrations of N-1-naphthylphthalamic acid (NPA) and verapamil, respectively, are required to inhibit translocation by 50% at simulated sunrise compared to mid-day. Gas chromatographic analysis displayed that ethylene evolution in A. palmeri was higher when dicamba was applied during mid-day compared to sunrise. Furthermore, it was found that inhibition of translocation via 2,3,5-triiodobenzoic acid (TIBA) resulted in an increased amount of 2,4-D-induced ethylene evolution at sunrise, and the inhibition of dicamba translocation via NPA reversed the difference in ethylene evolution across time of application. Dawn applications of these herbicides were associated with increased expression of a putative 9-cis-epoxycarotenoid dioxygenase biosynthesis gene NCED1, while there was a notable lack of trends observed across times of day and across herbicides with ACS1, encoding 1-aminocyclopropane-1-carboxylic acid synthase. Overall, this research indicates that translocation is differentially regulated via specific protein-level mechanisms across times of application, and that ethylene release, a chief phytotoxic process involved in the response to auxinic herbicides, is related to translocation. Furthermore, transcriptional regulation of abscisic acid involvement in phytotoxicity and/or translocation are suggested.

The Arabidopsis ATP-BINDING CASSETTE Transporter ABCB21 Regulates Auxin Levels in Cotyledons, the Root Pericycle, and Leaves.

The phytohormone auxin plays significant roles in regulating plant growth and development. In Arabidopsis, a subset of ATP-BINDING CASSETTE subfamily B (ABCB) transporters participate in polar movement of auxin by exclusion from and prevention of reuptake at the plasma membrane. A previous analysis identified ABCB21 as a conditional auxin uptake/efflux transporter that regulates cellular auxin levels, but clear physiological roles for ABCB21 in planta remain unknown. Here we show that ABCB21 maintains the acropetal auxin transport stream by regulating auxin levels in the pericycle. Loss of ABCB21 reduces rootward auxin transport and delays lateral root emergence. In seedling shoots, ABCB21 regulates mobilization of auxin from the photosynthetic cotyledons that is important for phototropic bending. In rosette leaves ABCB21 contributes to lateral auxin distribution. These results support a primary role for ABCB21 in regulating auxin distribution supplementary to the primary ABCB auxin transporters ABCB1 and 19.

Increased chalcone synthase (CHS) expression is associated with dicamba resistance in Kochia scoparia.

Resistance to the synthetic auxin herbicide dicamba is increasingly problematic in Kochia scoparia. The resistance mechanism in an inbred dicamba-resistant K. scoparia line (9425R) was investigated using physiological and transcriptomics (RNA-Seq) approaches. No differences were found in dicamba absorption or metabolism between 9425R and a dicamba-susceptible line, but 9425R was found to have significantly reduced dicamba translocation. Known auxin-responsive genes ACC synthase (ACS) and indole-3-acetic acid amino synthetase (GH3) were transcriptionally induced following dicamba treatment in dicamba-susceptible K. scoparia but not in 9425R. Chalcone synthase (CHS), the gene regulating synthesis of the flavonols quertecin and kaemperfol, was found to have twofold higher transcription in 9425R both without and 12 h after dicamba treatment. Increased CHS transcription co-segregated with dicamba resistance in a forward genetics screen using an F2 population. Prior work has shown that the flavonols quertecin and kaemperfol compete with auxin for intercellular movement and vascular loading via ATP-binding cassette subfamily B (ABCB) membrane transporters. The results of this study support a model in which constitutively increased CHS expression in the meristem produces more flavonols that would compete with dicamba for intercellular transport by ABCB transporters, resulting in reduced dicamba translocation. © 2017 Society of Chemical Industry.

Learning from each other: ABC transporter regulation by protein phosphorylation in plant and mammalian systems.

The ABC (ATP-binding cassette) transporter family in higher plants is highly expanded compared with those of mammalians. Moreover, some members of the plant ABC subfamily B (ABCB) display very high substrate specificity compared with their mammalian counterparts that are often associated with multi-drug resistance phenomena. In this review, we highlight prominent functions of plant and mammalian ABC transporters and summarize our knowledge on their post-transcriptional regulation with a focus on protein phosphorylation. A deeper comparison of regulatory events of human cystic fibrosis transmembrane conductance regulator (CFTR) and ABCB1 from the model plant Arabidopsis reveals a surprisingly high degree of similarity. Both physically interact with orthologues of the FK506-binding proteins that chaperon both transporters to the plasma membrane in an action that seems to involve heat shock protein (Hsp)90. Further, both transporters are phosphorylated at regulatory domains that connect both nt-binding folds. Taken together, it appears that ABC transporters exhibit an evolutionary conserved but complex regulation by protein phosphorylation, which apparently is, at least in some cases, tightly connected with protein-protein interactions (PPI).

Role of the N-terminal transmembrane domain in the endo-lysosomal targeting and function of the human ABCB6 protein.

ATP-binding cassette, subfamily B (ABCB) 6 is a homodimeric ATP-binding cassette (ABC) transporter present in the plasma membrane and in the intracellular organelles. The intracellular localization of ABCB6 has been a matter of debate, as it has been suggested to reside in the mitochondria and the endo-lysosomal system. Using a variety of imaging modalities, including confocal microscopy and EM, we confirm the endo-lysosomal localization of ABCB6 and show that the protein is internalized from the plasma membrane through endocytosis, to be distributed to multivesicular bodies and lysosomes. In addition to the canonical nucleotide-binding domain (NBD) and transmembrane domain (TMD), ABCB6 contains a unique N-terminal TMD (TMD0), which does not show sequence homology to known proteins. We investigated the functional role of these domains through the molecular dissection of ABCB6. We find that the folding, dimerization, membrane insertion and ATP binding/hydrolysis of the core–ABCB6 complex devoid of TMD0 are preserved. However, in contrast with the full-length transporter, the core–ABCB6 construct is retained at the plasma membrane and does not appear in Rab5-positive endosomes. TMD0 is directly targeted to the lysosomes, without passage to the plasma membrane. Collectively, our results reveal that TMD0 represents an independently folding unit, which is dispensable for catalysis, but has a crucial role in the lysosomal targeting of ABCB6.

Arabidopsis thaliana AUCSIA-1 Regulates Auxin Biology and Physically Interacts with a Kinesin-Related Protein

Aucsia is a green plant gene family encoding 44–54 amino acids long miniproteins. The sequenced genomes of most land plants contain two Aucsia genes. RNA interference of both tomato (Solanum lycopersicum) Aucsia genes (SlAucsia-1 and SlAucsia-2) altered auxin sensitivity, auxin transport and distribution; it caused parthenocarpic development of the fruit and other auxin-related morphological changes. Here we present data showing that the Aucsia-1 gene of Arabidopsis thaliana alters, by itself, root auxin biology and that the AtAUCSIA-1 miniprotein physically interacts with a kinesin-related protein. The AtAucsia-1 gene is ubiquitously expressed, although its expression is higher in roots and inflorescences in comparison to stems and leaves. Two allelic mutants for AtAucsia-1 gene did not display visible root morphological alterations; however both basipetal and acropetal indole-3-acetic acid (IAA) root transport was reduced as compared with wild-type plants. The transcript steady state levels of the auxin efflux transporters ATP BINDING CASSETTE subfamily B (ABCB) ABCB1, ABCB4 and ABCB19 were reduced in ataucsia-1 plants. In ataucsia-1 mutant, lateral root growth showed an altered response to i) exogenous auxin, ii) an inhibitor of polar auxin transport and iii) ethylene. Overexpression of AtAucsia-1 inhibited primary root growth. In vitro and in vivo protein-protein interaction experiments showed that AtAUCSIA-1 interacts with a 185 amino acids long fragment belonging to a 2712 amino acids long protein of unknown function (At4g31570). Bioinformatics analysis indicates that the AtAUCSIA-1 interacting protein (AtAUCSIA-1IP) clusters with a group of CENP-E kinesin-related proteins. Gene ontology predictions for the two proteins are consistent with the hypothesis that the AtAUCSIA-1/AtAUCSIA-1IP complex is involved in the regulation of the cytoskeleton dynamics underlying auxin biology.

655 Genetic Polymorphism in the Multi-Drug Resistance-5 Gene is Associated With Non-Response to Azathioprine Treatment in Inflammatory Bowel Disease

Introduction Multidrug resistance (MDR) genes encode transmembrane ATP-dependent pumps, otherwise known as the ATP-binding cassette subfamily-b (ABCB) transporters responsible for removing xenobiotics, particularly drugs, from cells. Thiopurine metabolites are exported from cells by MDR-4 and MDR-5. Altered ABCB pump activity has been linked to treatment resistance in other contexts. Methods Using Taqman® real-time PCR genotyping assays, a prospective cohort of 192 patients receiving azathioprine for IBD was genotyped for three coding region SNPs in MDR4 and three in MDR5, (all of which occur commonly in the Caucasian population). The SNPs tested were in MDR4: 175C→T, 711A→T and 1954A→G and in MDR5: 2T→C, 343A→G and 1573G→A. Association between genotype and clinical outcome on azathioprine treatment (complete response, non-response or withdrawal due to side-effects) was sought. Association tests were performed under a dominant model, using Fisher9s Exact test. No correction for multiple testing has been applied. Results The SNP MDR-5 c.343A→G was associated with a lack of clinical response to thiopurine treatment p=0.02, OR 2.43, 95%CI 1.14 to 5.17. Moreover, when analysed alongside other known markers of non-response to thiopurine treatment, (TPMT activity >35 pmol/h/mgHb and AOX 3404A→G) increasing numbers of markers increased their chance of non-response, p=0.0001 (χ2 for trend). No other SNP had a significant effect on clinical response and no SNP was associated with ADRs. Conclusion This study raises the possibility that MDR polymorphism could be important in thiopurine pharmacogenetics and warrants further study in other cohorts. Reliable markers of non-response to thiopurines could be incorporated into a panel of pharmacogenetic markers to inform the selection and dosing of immunomodulatory drugs, offering each individual the best chance of achieving early effective disease control Abstract 043.

PTU-043 Genetic polymorphism in the multidrug resistance-5 gene is associated with non-response to azathioprine treatment in inflammatory bowel disease: Abstract PTU-043

<h3>Introduction</h3> Multidrug resistance (MDR) genes encode transmembrane ATP-dependent pumps, otherwise known as the ATP-binding cassette subfamily-b (ABCB) transporters responsible for removing xenobiotics, particularly drugs, from cells. Thiopurine metabolites are exported from cells by MDR-4 and MDR-5. Altered ABCB pump activity has been linked to treatment resistance in other contexts. <h3>Methods</h3> Using Taqman® real-time PCR genotyping assays, a prospective cohort of 192 patients receiving azathioprine for IBD was genotyped for three coding region SNPs in MDR4 and three in MDR5, (all of which occur commonly in the Caucasian population). The SNPs tested were in MDR4: 175C→T, 711A→T and 1954A→G and in MDR5: 2T→C, 343A→G and 1573G→A. Association between genotype and clinical outcome on azathioprine treatment (complete response, non-response or withdrawal due to side-effects) was sought. Association tests were performed under a dominant model, using Fisher9s Exact test. No correction for multiple testing has been applied. <h3>Results</h3> The SNP MDR-5 c.343A→G was associated with a lack of clinical response to thiopurine treatment p=0.02, OR 2.43, 95%CI 1.14 to 5.17. Moreover, when analysed alongside other known markers of non-response to thiopurine treatment, (TPMT activity &gt;35 pmol/h/mgHb and AOX 3404A→G) increasing numbers of markers increased their chance of non-response, p=0.0001 (χ² for trend). No other SNP had a significant effect on clinical response and no SNP was associated with ADRs. <h3>Conclusion</h3> This study raises the possibility that MDR polymorphism could be important in thiopurine pharmacogenetics and warrants further study in other cohorts. Reliable markers of non-response to thiopurines could be incorporated into a panel of pharmacogenetic markers to inform the selection and dosing of immunomodulatory drugs, offering each individual the best chance of achieving early effective disease control Abstract 043.