Behavioral and Metabolic Effects of ABCG4 KO in the APPswe,Ind (J9) Mouse Model of Alzheimer's Disease.

The pathogenesis of Alzheimer's disease (AD) is complex and involves an imbalance between production and clearance of amyloid-ß peptides (Aß), resulting in accumulation of Aß in senile plaques. Hypercholesterolemia is a major risk factor for developing AD, with cholesterol shown to accumulate in senile plaques and increase production of Aß. ABCG4 is a member of the ATP-binding cassette transporters predominantly expressed in the CNS and has been suggested to play a role in cholesterol and Aß efflux from the brain. In this study, we bred Abcg4 knockout (KO) with the APPSwe,Ind (J9) mouse model of AD to test the hypothesis that loss of Abcg4 would exacerbate the AD phenotype. Unexpectedly, no differences were observed in novel object recognition (NOR) and novel object placement (NOP) behavioral tests, or on histologic examinations of brain tissues for senile plaque numbers. Furthermore, clearance of radiolabeled Aß from the brains did not differ between Abcg4 KO and control mice. Metabolic testing by indirect calorimetry, glucose tolerance test (GTT), and insulin tolerance test (ITT) were also mostly similar between groups with only a few mild metabolic differences noted. Overall, these data suggest that the loss of ABCG4 did not exacerbate the AD phenotype.

Abscisic acid root-to-shoot translocation by transporter AtABCG25 mediates stomatal movements in Arabidopsis.

The phytohormone abscisic acid (ABA) plays a central role in regulating stomatal movements under drought conditions. The root-derived peptide CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 25 (CLE25) moves from the root to shoot for activating ABA biosynthesis under drought conditions. However, the root-to-shoot translocation of root-derived ABA and its regulation of stomatal movements in the shoot remain to be clarified. Here, we reveal that the ABA transporter ATP-binding cassette subfamily G member 25 (AtABCG25) mediates root-to-shoot translocation of ABA and ABA-glucosyl ester (ABA-GE) in Arabidopsis (Arabidopsis thaliana). Isotope-labeled ABA tracer experiments and hormone quantification in xylem sap showed that the root-to-shoot translocation of ABA and ABA-GE was substantially impaired in the atabcg25 mutant under nondrought and drought conditions. However, the contents of ABA and ABA-GE in the leaves were lower in the atabcg25 mutant than in the wild type (WT) under nondrought but similar under drought conditions. Consistently, the stomatal closure was suppressed in the atabcg25 mutant under nondrought but not under drought conditions. The transporter activity assays showed that AtABCG25 directly exported ABA and ABA-GE in planta and in yeast (Saccharomyces cerevisiae) cells. Thus, we proposed a working model in which root-derived ABA transported by AtABCG25 via xylem mediates stomatal movements in the shoot under nondrought conditions but might exhibit little effect on stomatal movements under drought conditions. These findings extend the functions of AtABCG25 and provide insights into the long-distance translocation of ABA and its role in stomatal movements.

Endomembrane-biased dimerization of ABCG16 and ABCG25 transporters determines their substrate selectivity in ABA-regulated plant growth and stress responses.

ATP-binding cassette (ABC) transporters are integral membrane proteins that have evolved diverse functions fulfilled via the transport of various substrates. In Arabidopsis, the G subfamily of ABC proteins is particularly abundant and participates in multiple signaling pathways during plant development and stress responses. In this study, we revealed that two Arabidopsis ABCG transporters, ABCG16 and ABCG25, engage in ABA-mediated stress responses and early plant growth through endomembrane-specific dimerization-coupled transport of ABA and ABA-glucosyl ester (ABA-GE), respectively. We first revealed that ABCG16 contributes to osmotic stress tolerance via ABA signaling. More specifically, ABCG16 induces cellular ABA efflux in both yeast and plant cells. Using FRET analysis, we showed that ABCG16 forms obligatory homodimers for ABA export activity and that the plasma membrane-resident ABCG16 homodimers specifically respond to ABA, undergoing notable conformational changes. Furthermore, we demonstrated that ABCG16 heterodimerizes with ABCG25 at the endoplasmic reticulum (ER) membrane and facilitates the ER entry of ABA-GE in both Arabidopsis and tobacco cells. The specific responsiveness of the ABCG16-ABCG25 heterodimer to ABA-GE and the superior growth of their double mutant support an inhibitory role of these two ABCGs in early seedling establishment via regulation of ABA-GE translocation across the ER membrane. Our endomembrane-specific analysis of the FRET signals derived from the homo- or heterodimerized ABCG complexes allowed us to link endomembrane-biased dimerization to the translocation of distinct substrates by ABCG transporters, providing a prototypic framework for understanding the omnipotence of ABCG transporters in plant development and stress responses.

High cholesterol absorption: A risk factor of atherosclerotic cardiovascular diseases?

Lowering elevated low-density lipoprotein cholesterol (LDL-C) concentrations reduces the risk of atherosclerotic cardiovascular diseases (ASCVDs). However, increasing evidence suggests that cholesterol metabolism may also be involved in the risk reduction of ASCVD events. In this review, we discuss if the different profiles of cholesterol metabolism, with a focus on high cholesterol absorption, are atherogenic, and what could be the possible mechanisms. The potential associations of cholesterol metabolism and the risk of ASCVDs are evaluated from genetic, metabolic, and population-based studies and lipid-lowering interventions. According to these studies, loss-of-function genetic variations in the small intestinal sterol transporters ABCG5 and ABCG8 result in high cholesterol absorption associated with low cholesterol synthesis, low cholesterol elimination from the body, and a high risk of ASCVDs. In contrast, loss-of-function genetic variations in another intestinal sterol transporter, NPC1L1 result in low cholesterol absorption associated with high cholesterol synthesis, elevated cholesterol elimination from the body, and low risk of ASCVDs. Statin monotherapy is not sufficient to reduce the ASCVD risk in cases of high cholesterol absorption, and these individuals need combination therapy of statin with cholesterol absorption inhibition. High cholesterol absorption, i.e., >60%, is estimated to occur in approximately one third of a population, so taking it into consideration is important to optimise lipid-lowering therapy to prevent atherosclerosis and reduce the risk of ASCVD events.

An ABCG-type transporter intensifies ametryn catabolism by Phase III reaction mechanism in rice.

Pesticide residues in food crops are one of the seriously environmental contaminants that risk food safety and human health. Understanding the mechanism for pesticide catabolism is critical to develop effective biotechniques for rapid eliminating the residues in food crops. In this study we characterized a novel ABC transporter family gene ABCG52 (PDR18) in regulating rice response to pesticide ametryn (AME) widely used in the farmland. Efficient biodegradation of AME was evaluated by measuring its biotoxicity, accumulation, and metabolites in rice plants. OsPDR18 was localized to the plasma membrane and strongly induced under AME exposure. Transgenic rice overexpressing OsPDR18 (OE) conferred rice resistance and detoxification to AME by increasing chlorophyll contents, improving growth phenotypes, and reducing AME accumulation in plants. The AME concentrations in OE plants were only 71.8-78.1% (shoots) and 75.0-83.3% (roots) of the wild type. Mutation of OsPDR18 by CRISPR/Cas9 protocol led to the compromised growth and enhanced AME accumulation in rice. Five AME metabolites for Phase I and 13 conjugates for Phase II reactions in rice were characterized by HPLC/Q-TOF-HRMS/MS. Relative content analysis revealed that the AME metabolic products in OE plants were significantly reduced compared with wild-type. Importantly, the OE plants accumulated less AME metabolites and conjugates in rice grains, suggesting that OsPDR18 expression may actively facilitate the transport of AME for catabolism. These data unveil a AME catabolic mechanism by which OsPDR18 contributes to the AME detoxification and degradation in rice crops.

Restriction of access to the central cavity is a major contributor to substrate selectivity in plant ABCG transporters.

ABCG46 of the legume Medicago truncatula is an ABC-type transporter responsible for highly selective translocation of the phenylpropanoids, 4-coumarate, and liquiritigenin, over the plasma membrane. To investigate molecular determinants of the observed substrate selectivity, we applied a combination of phylogenetic and biochemical analyses, AlphaFold2 structure prediction, molecular dynamics simulations, and mutagenesis. We discovered an unusually narrow transient access path to the central cavity of MtABCG46 that constitutes an initial filter responsible for the selective translocation of phenylpropanoids through a lipid bilayer. Furthermore, we identified remote residue F562 as pivotal for maintaining the stability of this filter. The determination of individual amino acids that impact the selective transport of specialized metabolites may provide new opportunities associated with ABCGs being of interest, in many biological scenarios.

Triphasic regulation of ZmMs13 encoding an ABCG transporter is sequentially required for callose dissolution, pollen exine and anther cuticle formation in maize.

INTRODUCTION: ATP Binding Cassette G (ABCG) transporters are associated with plant male reproduction, while their regulatory mechanisms underlying anther and pollen development remain largely unknown. OBJECTIVES: Identify and characterize a male-sterility gene ZmMs13 encoding an ABCG transporter in modulating anther and pollen development in maize. METHODS: Phenotypic, cytological observations, and histochemistry staining were performed to characterize the ms13-6060 mutant. Map-based cloning and CRISPR/Cas9 gene editing were used to identify ZmMs13 gene. RNA-seq data and qPCR analyses, phylogenetic and microsynteny analyses, transient dual-luciferase reporter and EMSA assays, subcellular localization, and ATPase activity and lipidomic analyses were carried out to determine the regulatory mechanisms of ZmMs13 gene. RESULTS: Maize ms13-6060 mutant displays complete male sterility with delayed callose degradation, premature tapetal programmed cell death (PCD), and defective pollen exine and anther cuticle formation. ZmMs13 encodes a plasm membrane (PM)- and endoplasmic reticulum (ER)-localized half-size ABCG transporter (ZmABCG2a). The allele of ZmMs13 in ms13-6060 mutant has one amino acid (I311) deletion due to a 3-bp deletion in its fourth exon. The I311 and other conserved amino acid K99 are essential for the ATPase and lipid binding activities of ZmMS13. ZmMs13 is specifically expressed in anthers with three peaks at stages S5, S8b, and S10, which are successively regulated by transcription factors ZmbHLH122, ZmMYB84, and ZmMYB33-1/-2 at these three stages. The triphasic regulation of ZmMs13 is sequentially required for callose dissolution, tapetal PCD and pollen exine development, and anther cuticle formation, corresponding to transcription alterations of callose-, ROS-, PCD-, sporopollenin-, and anther cuticle-related genes in ms13-6060 anthers. CONCLUSION: ms13-6060 mutation with one key amino acid (I311) deletion greatly reduces ZmMS13 ATPase and lipid binding activities and displays multiple effects during maize male reproduction. Our findings provide new insights into molecular mechanisms of ABCG transporters controlling anther and pollen development and male fertility in plants.

Characterization of the ABC Transporter G Subfamily in Pomegranate and Function Analysis of PgrABCG14.

ATP-binding cassette subfamily G (ABCG) proteins play important roles in plant growth and development by transporting metabolites across cell membranes. To date, the genetic characteristics and potential functions of pomegranate ABCG proteins (PgrABCGs) have remained largely unknown. In this study, we found that 47 PgrABCGs were divided into five groups according to a phylogenetic analysis; groups I, II, III, and IV members are half-size proteins, and group V members are full-size proteins. PgrABCG14, PgrABCG21, and PgrABCG47 were highly expressed in the inner seed coat but had very low expression levels in the outer seed coat, and the expression levels of these three PgrABCG genes in the inner seed coats of hard-seeded pomegranate 'Dabenzi' were higher than those of soft-seeded pomegranate 'Tunisia'. In addition, the expression of these three PgrABCG genes was highly correlated with the expression of genes involved in lignin biosynthesis and hormone signaling pathways. The evolution of PgrABCG14 presents a highly similar trend to the origin and evolution of lignin biosynthesis during land plant evolution. Ectopic expression of PgrABCG14 in Arabidopsis promoted plant growth and lignin accumulation compared to wild type plants; meanwhile, the expression levels of lignin biosynthesis-related genes (CAD5, C4H, and Prx71) and cytokinin response marker genes (ARR5 and ARR15) were significantly upregulated in transgenic plants, which suggests the potential role of PgrABCG14 in promoting plant growth and lignin accumulation. Taken together, these findings not only provide insight into the characteristics and evolution of PgrABCGs, but also shed a light on the potential functions of PgrABCGs in seed hardness development.

ATP-Binding Cassette G Transporters and Their Multiple Roles Especially for Male Fertility in Arabidopsis, Rice and Maize.

ATP-binding cassette subfamily G (ABCG) transporters are extensive in plants and play essential roles in various processes influencing plant fitness, but the research progress varies greatly among Arabidopsis, rice and maize. In this review, we present a consolidated nomenclature and characterization of the whole 51 ABCG transporters in maize, perform a phylogenetic analysis and classification of the ABCG subfamily members in maize, and summarize the latest research advances in ABCG transporters for these three plant species. ABCG transporters are involved in diverse processes in Arabidopsis and rice, such as anther and pollen development, vegetative and female organ development, abiotic and biotic stress response, and phytohormone transport, which provide useful clues for the functional investigation of ABCG transporters in maize. Finally, we discuss the current challenges and future perspectives for the identification and mechanism analysis of substrates for plant ABCG transporters. This review provides a basic framework for functional research and the potential application of ABCG transporters in multiple plants, including maize.

Modes of secretion of plant lipophilic metabolites via ABCG transporter-dependent transport and vesicle-mediated trafficking.

Many lipophilic metabolites produced by terrestrial plants are deposited on plant surfaces to protect them from abiotic and biotic stresses. Plant-derived lipophilic metabolites include apoplastic biopolymers, such as wax, cutin, sporopollenin, suberin, and lignin, as well as low-molecular-weight secondary metabolites. These secreted molecules confer adaptive toughness and robustness on plants. The mechanisms responsible for the secretion of these lipophilic metabolites remain unclear, although two pathways, mediated by transporters and vesicles, have been proposed. Recent genetic and biochemical studies have shown that G-type ATP-binding cassette (ABCG) transporters and membrane trafficking factors are involved in the apoplastic accumulation of lipophilic metabolites in plants. These two distinctive modes of secretion may be either exclusive or collaborative. This review describes these transporter-dependent and vesicle-mediated mechanisms underlying the secretion of lipophilic metabolites.

Knockout of ABC Transporter ABCG4 Gene Confers Resistance to Cry1 Proteins in Ostrinia furnacalis.

Ostrinia furnacalis is an important borer on maize. Long-term and large-scale planting of transgenic corn has led O. furnacalis evolving resistance and reducing the control effect. Recently, high levels of resistance to Bt Cry1 toxins have been reported to be genetically linked to the mutation or down-regulation of ABC transporter subfamily G gene ABCG4 in O. furnacalis. In order to further determine the relationship between ABCG4 gene and the resistance to Cry1 toxins in O. furnacalis, the novel CRISPR/Cas9 genome engineering system was utilized to successfully construct ABCG4-KO knockout homozygous strain. Bioassay results indicated that an ABCG4-KO strain had a higher resistance to Cry1 proteins compared with a susceptible strain (ACB-BtS). The result indicates that the ABCG4 gene may act as a receptor of the Bt Cry1 toxin in O. furnacalis. Furthermore, the development time was significantly changed in the early stage ABCG4-KO larvae, and the population parameters were also significantly changed. In summary, our CRISPR/Cas9-mediated genome editing study presents evidence that ABCG4 gene is a functional receptor for Bt Cry1 toxins, laying the foundation for further clarification of the Bt resistance mechanism.

Mutants of the white ABCG Transporter in Drosophila melanogaster Have Deficient Olfactory Learning and Cholesterol Homeostasis.

Drosophila's white gene encodes an ATP-binding cassette G-subfamily (ABCG) half-transporter. White is closely related to mammalian ABCG family members that function in cholesterol efflux. Mutants of white have several behavioral phenotypes that are independent of visual defects. This study characterizes a novel defect of white mutants in the acquisition of olfactory memory using the aversive olfactory conditioning paradigm. The w1118 mutants learned slower than wildtype controls, yet with additional training, they reached wildtype levels of performance. The w1118 learning phenotype is also found in the wapricot and wcoral alleles, is dominant, and is rescued by genomic white and mini-white transgenes. Reducing dietary cholesterol strongly impaired olfactory learning for wildtype controls, while w1118 mutants were resistant to this deficit. The w1118 mutants displayed higher levels of cholesterol and cholesterol esters than wildtype under this low-cholesterol diet. Increasing levels of serotonin, dopamine, or both in the white mutants significantly improved w1118 learning. However, serotonin levels were not lower in the heads of the w1118 mutants than in wildtype controls. There were also no significant differences found in synapse numbers within the w1118 brain. We propose that the w1118 learning defect may be due to inefficient biogenic amine signaling brought about by altered cholesterol homeostasis.

ZmFAR1 and ZmABCG26 Regulated by microRNA Are Essential for Lipid Metabolism in Maize Anther.

The function and regulation of lipid metabolic genes are essential for plant male reproduction. However, expression regulation of lipid metabolic genic male sterility (GMS) genes by noncoding RNAs is largely unclear. Here, we systematically predicted the microRNA regulators of 34 maize white brown complex members in ATP-binding cassette transporter G subfamily (WBC/ABCG) genes using transcriptome analysis. Results indicate that the ZmABCG26 transcript was predicted to be targeted by zma-miR164h-5p, and their expression levels were negatively correlated in maize B73 and Oh43 genetic backgrounds based on both transcriptome data and qRT-PCR experiments. CRISPR/Cas9-induced gene mutagenesis was performed on ZmABCG26 and another lipid metabolic gene, ZmFAR1. DNA sequencing, phenotypic, and cytological observations demonstrated that both ZmABCG26 and ZmFAR1 are GMS genes in maize. Notably, ZmABCG26 proteins are localized in the endoplasmic reticulum (ER), chloroplast/plastid, and plasma membrane. Furthermore, ZmFAR1 shows catalytic activities to three CoA substrates in vitro with the activity order of C12:0-CoA > C16:0-CoA > C18:0-CoA, and its four key amino acid sites were critical to its catalytic activities. Lipidomics analysis revealed decreased cutin amounts and increased wax contents in anthers of both zmabcg26 and zmfar1 GMS mutants. A more detailed analysis exhibited differential changes in 54 monomer contents between wild type and mutants, as well as between zmabcg26 and zmfar1. These findings will promote a deeper understanding of miRNA-regulated lipid metabolic genes and the functional diversity of lipid metabolic genes, contributing to lipid biosynthesis in maize anthers. Additionally, cosegregating molecular markers for ZmABCG26 and ZmFAR1 were developed to facilitate the breeding of male sterile lines.

Dynamic changes of phosphatidylinositol and phosphatidylinositol 4-phosphate levels modulate H+-ATPase and Na+/H+ antiporter activities to maintain ion homeostasis in Arabidopsis under salt stress.

Plant metabolites are dynamically modified and distributed in response to environmental changes. However, it is poorly understood how metabolic change functions in plant stress responses. Maintaining ion homeostasis under salt stress requires coordinated activation of two types of central regulators: plasma membrane (PM) H+-ATPase and Na+/H+ antiporter. In this study, we used a bioassay-guided isolation approach to identify endogenous small molecules that affect PM H+-ATPase and Na+/H+ antiporter activities and identified phosphatidylinositol (PI), which inhibits PM H+-ATPase activity under non-stress conditions in Arabidopsis by directly binding to the C terminus of the PM H+-ATPase AHA2. Under salt stress, the phosphatidylinositol 4-phosphate-to-phosphatidylinositol (PI4P-to-PI) ratio increased, and PI4P bound and activated the PM Na+/H+ antiporter. PI prefers binding to the inactive form of PM H+-ATPase, while PI4P tends to bind to the active form of the Na+/H+ antiporter. Consistent with this, pis1 mutants, with reduced levels of PI, displayed increased PM H+-ATPase activity and salt stress tolerance, while the pi4kß1 mutant, with reduced levels of PI4P, displayed reduced PM Na+/H+ antiporter activity and salt stress tolerance. Collectively, our results reveal that the dynamic change between PI and PI4P in response to salt stress in Arabidopsis is crucial for maintaining ion homeostasis to protect plants from unfavorable environmental conditions.

ABCG1 and ABCG4 as key transporters in the development of pulmonary alveolar proteinosis by nanoparticles.

Pulmonary alveolar proteinosis (PAP) has been reported in rodents treated with nanoparticles (NPs). However, little is known about the type of NPs producing PAP and their toxicity mechanisms. Here, we assembled seven PAP-inducing NPs and TiO2 NPs as a negative control. At 1 and 6 months after a single intratracheal instillation in rats, pulmonary inflammation and the gene expression of ATP-binding cassette (ABC) transporters and related genes were evaluated in separated alveolar macrophages (AMs). One month after intratracheal instillation, seven NPs (Eu2O3, In2O3, Pr6O11, Sm2O3, Tb4O7, and NiO) caused PAP, but only In2O3 NPs caused persistent PAP at 6 months after treatment. The levels of phospholipids, indicators of PAP, showed good correlations with the gene expression profile of five transporters (ABCA1, ABCB4, ABCB8, ABCG1, and ABCG4), which effluxing phospholipids in AMs. Among them, ABCG1 and ABCG4 might be key transporters involved in PAP development because both showed a negative correlation with the magnitude of PAP, while others might be compensatory transporters for PAP recovery, as they showed a positive correlation. In conclusion, the identification of seven PAP-producing NPs implies that PAP may be an emerging occupational disease and that ABCG1 and ABCG4 may be therapeutic targets for PAP.

ESCRT components ISTL1 andLIP5 are required for tapetal function and pollen viability.

Pollen wall assembly is crucial for pollen development and plant fertility. The durable biopolymer sporopollenin and the constituents of the tryphine coat are delivered to developing pollen grains by the highly coordinated secretory activity of the surrounding tapetal cells. The role of membrane trafficking in this process, however, is largely unknown. In this study, we used Arabidopsis thaliana to characterize the role of two late-acting endosomal sorting complex required for transport (ESCRT) components, ISTL1 and LIP5, in tapetal function. Plants lacking ISTL1 and LIP5 form pollen with aberrant exine patterns, leading to partial pollen lethality. We found that ISTL1 and LIP5 are required for exocytosis of plasma membrane and secreted proteins in the tapetal cells at the free microspore stage, contributing to pollen wall development and tryphine deposition. Whereas the ESCRT machinery is well known for its role in endosomal trafficking, the function of ISTL1 and LIP5 in exocytosis is not a typical ESCRT function. The istl1 lip5 double mutants also show reduced intralumenal vesicle concatenation in multivesicular endosomes in both tapetal cells and developing pollen grains as well as morphological defects in early endosomes/trans-Golgi networks, suggesting that late ESCRT components function in the early endosomal pathway and exocytosis.

Identification of ABCG transporter genes associated with chlorantraniliprole resistance in Plutella xylostella (L.).

BACKGROUND: Plutella xylostella (L.) is a serious worldwide pest that feeds on cruciferous plants and has evolved resistance to different classes of insecticides used for its control, including chlorantraniliprole. ATP-binding cassette (ABC) transporters, constituting the largest transport family in organisms, are involved in phase III of the detoxification process and may play important roles in insecticide resistance. RESULTS: A total of 15 ABC transporter transcripts from subfamily G were identified in P. xylostella based on the latest DBM genome. Synergism studies showed that treatment with verapamil, a potent inhibitor of ABC transporters, significantly increased the toxicity of chlorantraniliprole against larvae of two chlorantraniliprole-resistant P. xylostella populations (NIL and BL). ABCG2, ABCG5, ABCG6, ABCG9, ABCG11, ABCG14 and ABCG15 were significantly overexpressed in NIL and BL compared with the susceptible population (SS), and ABCG1, ABCG6, ABCG8, ABCG9, ABCG14 and ABCG15 were significantly upregulated after treatment with the LC50 of chlorantraniliprole in SS. Subsequently, ABCG6, ABCG9 and ABCG14, which were overexpressed in both NIL and BL and could be induced in SS, were chosen for functional study. RNAi-mediated knockdown of each of the three ABCGs significantly increased the sensitivity of larvae to chlorantraniliprole. These results confirmed that overexpression of ABCG6, ABCG9 and ABCG14 may contribute to chlorantraniliprole resistance in P. xylostella. CONCLUSION: Overexpression of some genes in the ABCG subfamily is involved in P. xylostella resistance to chlorantraniliprole. These results may help to establish a foundation for further studies investigating the role played by ABC transporters in chlorantraniliprole resistance in P. xylostella or other insect pests. © 2021 Society of Chemical Industry.

ABCG transporters export cutin precursors for the formation of the plant cuticle.

The plant cuticle is deposited on the surface of primary plant organs, such as leaves, fruits, and floral organs, forming a diffusion barrier and protecting the plant against various abiotic and biotic stresses. Cutin, the structural polyester of the plant cuticle, is synthesized in the apoplast. Plasma-membrane-localized ATP-binding cassette (ABC) transporters of the G family have been hypothesized to export cutin precursors. Here, we characterize SlABCG42 of tomato representing an ortholog of AtABCG32 in Arabidopsis. SlABCG42 expression in Arabidopsis complements the cuticular deficiencies of the Arabidopsis pec1/abcg32 mutant. RNAi-dependent downregulation of both tomato genes encoding proteins highly homologous to AtABCG32 (SlABCG36 and SlABCG42) leads to reduced cutin deposition and formation of a thinner cuticle in tomato fruits. By using a tobacco (Nicotiana benthamiana) protoplast system, we show that AtABCG32 and SlABCG42 have an export activity for 10,16-dihydroxy hexadecanoyl-2-glycerol, a cutin precursor in vivo. Interestingly, also free ω-hydroxy hexadecanoic acid as well as hexadecanedioic acid were exported, furthering the research on the identification of cutin precursors in vivo and the respective mechanisms of their integration into the cutin polymer.

Early stages of legume-rhizobia symbiosis are controlled by ABCG-mediated transport of active cytokinins.

Growing evidence has highlighted the essential role of plant hormones, notably, cytokinins (CKs), in nitrogen-fixing symbiosis, both at early and late nodulation stages1,2. Despite numerous studies showing the central role of CK in nodulation, the importance of CK transport in the symbiosis is unknown. Here, we show the role of ABCG56, a full-size ATP-binding cassette (ABC) transporter in the early stages of the nodulation. MtABCG56 is expressed in roots and nodules and its messenger RNA levels increase upon treatment with symbiotic bacteria, isolated Nod factor and CKs, accumulating within the epidermis and root cortex. MtABCG56 exports bioactive CKs in an ATP-dependent manner over the plasma membrane and its disruption results in an impairment of nodulation. Our data indicate that ABCG-mediated cytokinin transport is important for proper establishment of N-fixing nodules.

Bundle sheath suberisation is required for C4 photosynthesis in a Setaria viridis mutant.

C4 photosynthesis provides an effective solution for overcoming the catalytic inefficiency of Rubisco. The pathway is characterised by a biochemical CO2 concentrating mechanism that operates across mesophyll and bundle sheath (BS) cells and relies on a gas tight BS compartment. A screen of a mutant population of Setaria viridis, an NADP-malic enzyme type C4 monocot, generated using N-nitroso-N-methylurea identified a mutant with an amino acid change in the gene coding region of the ABCG transporter, a step in the suberin synthesis pathway. Here, Nile red staining, TEM, and GC/MS confirmed the alteration in suberin deposition in the BS cell wall of the mutant. We show that this has disrupted the suberin lamellae of BS cell wall and increased BS conductance to CO2 diffusion more than two-fold in the mutant. Consequently, BS CO2 partial pressure is reduced and CO2 assimilation was impaired in the mutant. Our findings provide experimental evidence that a functional suberin lamellae is an essential anatomical feature for efficient C4 photosynthesis in NADP-ME plants like S. viridis and have implications for engineering strategies to ensure future food security.