Maize PIMT2 repairs damaged 3-METHYLCROTONYL COA CARBOXYLASE in mitochondria, affecting seed vigor.

PROTEIN l-ISOASPARTYL O-METHYLTRANSFERASE (PIMT) affects seed vigor by repairing damaged proteins. While PIMT is capable of isoaspartyl (isoAsp) repair in all proteins, those proteins most susceptible to isoAsp formation have not been well characterized, and the mechanisms by which PIMT affects seed vigor remain largely unknown. Using co-immunoprecipitation and LC-MS/MS, we found that maize (Zea mays) PIMT2 (ZmPIMT2) interacted predominantly with both subunits of maize 3-METHYLCROTONYL COA CARBOXYLASE (ZmMCC). ZmPIMT2 is specifically expressed in the maize embryo. Both mRNA and protein levels of ZmPIMT2 increased during seed maturation and declined during imbibition. Maize seed vigor was decreased in the zmpimt2 mutant line, while overexpression of ZmPIMT2 in maize and Arabidopsis thaliana increased seed vigor upon artificial aging. ZmPIMT2 was localized in the mitochondria, as determined by subcellular localization assays using maize protoplasts. ZmPIMT2 binding to ZmMCCα was confirmed by luciferase complementation tests in both tobacco (Nicotiana benthamiana) leaves and maize protoplasts. Knockdown of ZmMCCα decreased maize seed aging tolerance. Furthermore, overexpression of ZmPIMT2 decreased the accumulation of isoAsp of ZmMCCα protein in seed embryos that underwent accelerated aging treatment. Taken together, our results demonstrate that ZmPIMT2 binds ZmMCCα in mitochondria, repairs isoAsp damage, and positively affects maize seed vigor.

Raffinose positively regulates maize drought tolerance by reducing leaf transpiration.

Drought stress is one of the major constraints of global crop production. Raffinose, a non-reducing trisaccharide, has been considered to regulate positively the plant drought stress tolerance; however, evidence that augmenting raffinose production in leaves results in enhanced plant drought stress tolerance is lacking. The biochemical mechanism through which raffinose might act to mitigate plant drought stress remains unidentified. ZmRAFS encodes Zea mays RAFFINOSE SYNTHASE, a key enzyme that transfers galactose from the galactoside galactinol to sucrose for raffinose production. Overexpression of ZmRAFS in maize increased the RAFS protein and the raffinose content and decreased the water loss of leaves and enhanced plant drought stress tolerance. The biomass of the ZmRAFS overexpressing plants was similar to that of non-transgenic control plants when grown under optimal conditions, but was significantly greater than that of non-transgenic plants when grown under drought stress conditions. In contrast, the percentage of water loss of the detached leaves from two independent zmrafs mutant lines, incapable of synthesizing raffinose, was greater than that from null segregant controls and this phenomenon was partially rescued by supplementation of raffinose to detached zmrafs leaves. In addition, while there were differences in water loss among different maize lines, there was no difference in stomata density or aperture. Taken together, our work demonstrated that overexpression of the ZmRAFS gene in maize, in contrast to Arabidopsis, increased the raffinose content in leaves, assisted the leaf to retain water, and enhanced the plant drought stress tolerance without causing a detectable growth penalty.

Raffinose catabolism enhances maize waterlogging tolerance by stimulating adventitious root growth and development.

Raffinose mitigates plant heat-, drought- and cold- stresses; however, whether raffinose contributes to plant waterlogging tolerance is unknown. The maize zmrafs-1 mutant seedlings lacking raffinose, generate fewer and shorter adventitious root (AR) and are more sensitive to waterlogging stress, while overexpression of ZmRAFS increases raffinose content, stimulates AR formation, and enhances the waterlogging tolerance of maize seedlings. Transcriptome analysis of NS (Null segregant) seedlings compared with that of zmrafs-1, particularly when waterlogged, revealed that the expression of genes related to galactose metabolism and the auxin biosynthetic pathway were upregulated by raffinose. Additionally, Indole-3-acetic acid (IAA) amounts significantly decreased or increased in zmrafs-1 or ZmRAFS-overexpressing seedlings, respectively. Inhibition of the hydrolysis of raffinose by DGJ (1-deoxygalactonojirimycin) decreased the waterlogging tolerance of maize seedlings, decreased the expression of genes encoding proteins related to auxin transport-related genes as well as the IAA level in the seedlings, suggesting that the hydrolysis of raffinose is necessary for maize waterlogging tolerance. These data demonstrate that raffinose catabolism stimulates adventitious root formation via auxin signaling pathway to enhance maize waterlogging tolerance.

The basic helix-loop-helix transcription factor gene, OsbHLH38, plays a key role in controlling rice salt tolerance.

The plant hormone abscisic acid (ABA) is crucial for plant seed germination and abiotic stress tolerance. However, the association between ABA sensitivity and plant abiotic stress tolerance remains largely unknown. In this study, 436 rice accessions were assessed for their sensitivity to ABA during seed germination. The considerable diversity in ABA sensitivity among rice germplasm accessions was primarily reflected by the differentiation between the Xian (indica) and Geng (japonica) subspecies and between the upland-Geng and lowland-Geng ecotypes. The upland-Geng accessions were most sensitive to ABA. Genome-wide association analyses identified four major quantitative trait loci containing 21 candidate genes associated with ABA sensitivity of which a basic helix-loop-helix transcription factor gene, OsbHLH38, was the most important for ABA sensitivity. Comprehensive functional analyses using knockout and overexpression transgenic lines revealed that OsbHLH38 expression was responsive to multiple abiotic stresses. Overexpression of OsbHLH38 increased seedling salt tolerance, while knockout of OsbHLH38 increased sensitivity to salt stress. A salt-responsive transcription factor, OsDREB2A, interacted with OsbHLH38 and was directly regulated by OsbHLH38. Moreover, OsbHLH38 affected rice abiotic stress tolerance by mediating the expression of a large set of transporter genes of phytohormones, transcription factor genes, and many downstream genes with diverse functions, including photosynthesis, redox homeostasis, and abiotic stress responsiveness. These results demonstrated that OsbHLH38 is a key regulator in plant abiotic stress tolerance.

Raffinose synthase enhances drought tolerance through raffinose synthesis or galactinol hydrolysis in maize and Arabidopsis plants.

Raffinose and its precursor galactinol accumulate in plant leaves during abiotic stress. RAFFINOSE SYNTHASE (RAFS) catalyzes raffinose formation by transferring a galactosyl group of galactinol to sucrose. However, whether RAFS contributes to plant drought tolerance and, if so, by what mechanism remains unclear. In this study, we report that expression of RAFS from maize (or corn, Zea mays) (ZmRAFS) is induced by drought, heat, cold, and salinity stresses. We found that zmrafs mutant maize plants completely lack raffinose and hyper-accumulate galactinol and are more sensitive to drought stress than the corresponding null-segregant (NS) plants. This indicated that ZmRAFS and its product raffinose contribute to plant drought tolerance. ZmRAFS overexpression in Arabidopsis enhanced drought stress tolerance by increasing myo-inositol levels via ZmRAFS-mediated galactinol hydrolysis in the leaves due to sucrose insufficiency in leaf cells and also enhanced raffinose synthesis in the seeds. Supplementation of sucrose to detached leaves converted ZmRAFS from hydrolyzing galactinol to synthesizing raffinose. Taken together, we demonstrate that ZmRAFS enhances plant drought tolerance through either raffinose synthesis or galactinol hydrolysis, depending on sucrose availability in plant cells. These results provide new avenues to improve plant drought stress tolerance through manipulation of the raffinose anabolic pathway.

ZmDREB2A regulates ZmGH3.2 and ZmRAFS, shifting metabolism towards seed aging tolerance over seedling growth.

Seed aging tolerance and rapid seedling growth are important agronomic traits for crop production; however, how these traits are controlled at the molecular level remains largely unknown. The unaged seeds of two independent maize DEHYDRATION-RESPONSIVE ELEMENT-BINDING2A mutant (zmdreb2a) lines, with decreased expression of GRETCHEN HAGEN3.2 (ZmGH3.2, encoding indole-3-acetic acid [IAA] deactivating enzyme), and increased IAA in their embryo, produced longer seedling shoots and roots, than the null segregant (NS) controls. However, the zmdreb2a seeds, with decreased expression of RAFFINOSE SYNTHASE (ZmRAFS) and less raffinose in their embryo, exhibit decreased seed aging tolerance, than the NS controls. Overexpression of ZmDREB2A in maize protoplasts increased the expression of ZmGH3.2, ZmRAFS genes and that of a Rennila LUCIFERASE reporter (Rluc) gene, which was controlled by either the ZmGH3.2- or ZmRAFS-promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation assay quantitative polymerase chain reaction showed that ZmDREB2A directly binds to the DRE motif of the promoters of both ZmGH3.2 and ZmRAFS. Exogenous supplementation of IAA to the unaged, germinating NS seeds increased subsequent seedling growth making them similar to the zmdreb2a seedlings from unaged seeds. These findings provide evidence that ZmDREB2A regulates the longevity of maize seed by stimulating the production of raffinose while simultaneously acting to limit auxin-mediated cell expansion.

Maize HSFA2 and HSBP2 antagonistically modulate raffinose biosynthesis and heat tolerance in Arabidopsis.

Raffinose is thought to play an important role in plant tolerance of abiotic stress. We report here that maize HEAT SHOCK FACTOR A2 (ZmHSFA2) and HEAT SHOCK BINDING PROTEIN 2 (ZmHSBP2) physically interact with each other and antagonistically modulate expression of GALACTINOL SYNTHASE2 (ZmGOLS2) and raffinose biosynthesis in transformed maize protoplasts and Arabidopsis plants. Overexpression of ZmHSFA2 in Arabidopsis increased the expression of Arabidopsis AtGOLS1, AtGOLS2 and AtRS5 (RAFFINOSE SYNTHASE), increased the raffinose content in leaves and enhanced plant heat stress tolerance. Contrary to ZmHSFA2, overexpression of ZmHSBP2 in Arabidopsis decreased expression of AtGOLS1, AtGOLS2 and AtRS5, decreased the raffinose content in leaves and reduced plant heat stress tolerance. ZmHSFA2 and ZmHSBP2 also interact with their Arabidopsis counterparts AtHSBP and AtHSFA2 as determined using bimolecular fluorescence complementation assays. Furthermore, endogenous ZmHSBP2 and Rluc, controlled by the ZmHSBP2 promoter, are transcriptionally activated by ZmHSFA2 and inhibited by ZmHSBP2 in maize protoplasts. These findings provide insights into the transcriptional regulation of raffinose biosynthetic genes, and the tolerance their product confers to plant heat stress.

ZmDREB1A Regulates RAFFINOSE SYNTHASE Controlling Raffinose Accumulation and Plant Chilling Stress Tolerance in Maize.

Raffinose accumulation is positively correlated with plant chilling stress tolerance; however, the understanding of the function and regulation of raffinose metabolism under chilling stress remains in its infancy. RAFFINOSE SYNTHASE (RAFS) is the key enzyme for raffinose biosynthesis. In this study, we report that two independent maize (Zea mays) zmrafs mutant lines, in which raffinose was completely abolished, were more sensitive to chilling stress and their net photosynthetic product (total soluble sugars and starch) accumulation was significantly decreased compared with controls after chilling stress. A similar characterization of the maize dehydration responsive element (DRE)-binding protein 1A mutant (zmdreb1a) showed that ZmRAFS expression and raffinose content were significantly decreased compared with its control under chilling stress. Overexpression of maize ZmDREB1A in maize leaf protoplasts increased ZmDREB1A amounts, which consequently upregulated the expression of maize ZmRAFS and the Renilla LUCIFERASE (Rluc), which was controlled by the ZmRAFS promoter. Deletion of the single dehydration-responsive element (DRE) in the ZmRAFS promoter abolished ZmDREB1A's influence on Rluc expression, while addition of three copies of the DRE in the ZmRAFS promoter dramatically increased Rluc expression when ZmDREB1A was simultaneously overexpressed. Electrophoretic mobility shift assays and chromatin immunoprecipitation-quantitative PCR demonstrated that ZmDREB1A directly binds to the DRE motif in the promoter of ZmRAFS both in vitro and in vivo. These data demonstrate that ZmRAFS, which was directly regulated by ZmDREB1A, enhances both raffinose biosynthesis and plant chilling stress tolerance.

ZmGOLS2, a target of transcription factor ZmDREB2A, offers similar protection against abiotic stress as ZmDREB2A.

GALACTINOL SYNTHASE is the first committed enzyme in the raffinose biosynthetic pathway. We have previously characterized the maize (Zea mays) GALACTINOL SYNTHASE2 gene (ZmGOLS2) as abiotic stress induced. To further investigate the regulation of ZmGOLS2 gene expression, individual luciferase expression vectors,in which the luciferase gene was controlled by different lengths of the ZmGOLS2 promoter, were co-transfected into maize protoplasts with either a ZmDREB2A- or a GFP-expression vector. Over-expression of ZmDREB2A up-regulated both the expression of the luciferase gene controlled by the ZmGOLS2 promoter and the endogenous ZmGOLS2 gene in protoplasts. Only one of the two DRE elements in the ZmGOLS2 promoter was identified as necessary for this up-regulation. Expression vectors of GFP, ZmGOLS2 or ZmDREB2A were stably transformed into Arabidopsis. Expression of ZmDREB2A up-regulated the AtGOLS3 gene but only over-expression of ZmGOLS2 resulted in hyper-accumulation of galactinol and raffinose. Regardless, under drought-, heat shock-, high osmotic- or salinity-stress conditions, both the ZmGOLS2- and the ZmDREB2A- expressing plants had greater germination percentages, greater percentages of seedlings becoming autotropic, and/or greater survival percentages during/after stress than the control plants. Under normal growing conditions, transgenic Arabidopsis plants expressing the ZmGOLS2 gene had similar growth to that of untransformed wild type or GFP-expressing control plants, whereas ZmDREB2A over-expressing plants exhibited retarded growth relative to either of the controls. These data suggest that over-expression of ZmGOLS2, rather than the transcription factor ZmDREB2A, is a more practical target for generation of abiotic-stress tolerant crops.

HIV-1 Tat regulates the expression of the dcw operon and stimulates the proliferation of bacteria.

Infections of pathogenic bacteria are very common in acquired immunodeficiency syndrome (AIDS) patients. However, the biological effects of HIV-1 Tat on bacteria are incompletely understood. In this study, HIV-1 Tat was expressed in Escherichia coli and Pseudomonas aeruginosa (PA01) to investigate its biological effects on bacteria. Bacterial cells expressing either HIV-1 Tat1-86 (Tat1-86) or HIV-1 Tat1-72 (Tat1-72) grow significantly faster than those with either only an empty vector or an unrelated control (GFP or Rluc). Supplementation of purified HIV-1 Tat1-86 or Tat1-101 protein into bacterial culture medium stimulated the growth of both E. coli and PA01. The expression profile of certain cell division-associated genes, such as those in the division cell wall (dcw) operon (ftsA, ftsQ, ftsW and ftsZ), yafO and zipA, was altered in HIV-1 Tat1-86 expressing E. coli BL21(DE3). Furthermore, the expression of firefly luciferase (Fluc) reporter gene, when engineered for control by the dcw promoter and terminator, was enhanced by HIV-1 Tat in E. coli, confirming that HIV-1 Tat transcriptionally regulates the expression of the dcw operon. The finding that HIV-1 Tat stimulates bacterial growth whether it is produced intracellularly or applied extracellularly may have relevance for HIV patients who are highly susceptible to opportunistic bacterial infections. Contents category: Viruses -Retroviruses. The GenBank accession number for the sequence of HIV-1 Tat1-86 is AF324439.1.

Alterations in Carbohydrate Quantities in Freeze-Dried, Relative to Fresh or Frozen Maize Leaf Disks.

For various reasons, leaves are occasionally lyophilized prior to storage at -80 °C and preparing extracts. Soluble carbohydrate identity and quantity from maize leaf disks were ascertained in two separate years using anion exchange HPLC with pulsed electrochemical detection. Analyses were made from disks after freezing in liquid nitrogen with or without subsequent lyophilization (both years) or directly after removal from plants with or without lyophilization (only in the second year). By adding the lyophilizing step, galactose content consistently increased and, frequently, so did galactoglycerols. The source of the galactose increase with the added lyophilizing step was not due to metabolizing raffinose, as the raffinose synthase (rafs) null mutant leaves, which do not make that trisaccharide, also had a similar increase in galactose content with lyophilization. Apparently, the ester linkages attaching free fatty acids to galactoglycerolipids of the chloroplast are particularly sensitive to cleavage during lyophilization, resulting in increases in galactoglycerols. Regardless of the galactose source, a systematic error is introduced for carbohydrate (and, most likely, also chloroplast mono- or digalactosyldiacylglycerol) amounts when maize leaf samples are lyophilized prior to extraction. The recognition of lyophilization as a source of galactose increase provides a cautionary note for investigators of soluble carbohydrates.

The G671V variant of MRP1/ABCC1 links doxorubicin-induced acute cardiac toxicity to disposition of the glutathione conjugate of 4-hydroxy-2-trans-nonenal.

OBJECTIVE: Doxorubicin-induced acute cardiotoxicity is associated with the Gly671Val (G671V; rs45511401) variant of multidrug resistance-associated protein 1 (MRP1). Doxorubicin redox cycling causes lipid peroxidation and generation of the reactive electrophile, 4-hydroxy-2-trans-nonenal (HNE). Glutathione forms conjugates with HNE, yielding an MRP1 substrate, GS-HNE, whose intracellular accumulation can cause toxicity. METHODS: We established stable HEK293 cell lines overexpressing wild-type MRP1 (HEKMRP1), G671V (HEKG671V), and R433S (HEKR433S), a variant not associated with doxorubicin-induced cardiotoxicity and investigated the sensitivity of HEKG671V cells to doxorubicin and transport capacity of G671V toward GS-HNE. RESULTS: In ATP-dependent transport studies using plasma membrane-derived vesicles, the Vmax (pmol/min/mg) for GS-HNE transport was the lowest for G671V (69±4) and the highest for R433S (972±213) compared with wild-type MRP1 (416±22), whereas the Km values were 2.8±0.4, 6.0 or more, and 1.7±0.2 µmol/l, respectively. In cells, the doxorubicin IC50 (48 h) was not different in HEKMRP1 (463 nmol/l) versus HEKR433S (645 nmol/l), but this parameter was significantly lower in HEKG671V (181 nmol/l). HEKG671V retained significantly (approximately 20%) more, whereas HEKR433S retained significantly less intracellular doxorubicin than HEKMRP1. Similarly, HEKG671V cells treated with 1.5 µmol/l of doxorubicin for 24 h retained significantly more GS-HNE. In cells treated with 0.5 µmol/l of doxorubicin for 48 , glutathione and glutathione disulfide levels and the glutathione/glutathione disulfide ratio were significantly decreased in HEKG671V versus HEKMRP1; these values were similar in HEKR433S versus HEKMRP1. CONCLUSION: These data suggest that decreased MRP1-dependent GS-HNE efflux contributes to increased doxorubicin toxicity in HEKG671V and potentially in individuals carrying the G671V variant.

Long non-coding RNA MRPS30 divergent transcript can be detected in the cytoplasm of triple-negative breast cancer cells and is targeted by microRNA-130b.

Long non-coding RNA (lncRNA) MRPS30 divergent transcript (also known as BRCAT54) is recently reported to promote lung cancer. The involvement of BRCAT54 in triple-negative breast cancer (TNBC) is unknown. This study investigated the role of BRCAT54 in TNBC. The expression of BRCAT54 and microRNA(miR)-130b was detected by RT-qPCR. The subcellular location of BRCAT54 in TNBC cells was analyzed by nuclear fractionation assay. Overexpression of BRCAT54 and miR-130b was achieved in TNBC cells to explore the interaction between then. The role of BRCAT54 and miR-130b in TNBC cell proliferation was evaluated by BrdU assay. BRCAT54 was downregulated in TNBC, while miR-130b was upregulated in TNBC tissues. BRCAT54 and miR-130b were inversely correlated across both TNBC and normal tissues. BRCAT54 was detected in cytoplasm and was predicted to be targeted by miR-130b. In TNBC cells, downregulation of BRCAT54 was observed after the overexpression of miR-130b. Moreover, BRCAT54 decreased cell proliferation and miR-130b increased cell proliferation. Besides, BRCAT54 suppressed the role of miR-130b in increasing cell proliferation. Therefore, BRCAT54 can be detected in cytoplasm and was targeted by miR-130b to increase cell proliferation.

WATER-SOAKED SPOT1 Controls Chloroplast Development and Leaf Senescence via Regulating Reactive Oxygen Species Homeostasis in Rice.

Transmembrane kinases (TMKs) play important roles in plant growth and signaling cascades of phytohormones. However, its function in the regulation of early leaf senescence (ELS) of plants remains unknown. Here, we report the molecular cloning and functional characterization of the WATER-SOAKED SPOT1 gene which encodes a protein belongs to the TMK family and controls chloroplast development and leaf senescence in rice (Oryza sativa L.). The water-soaked spot1 (oswss1) mutant displays water-soaked spots which subsequently developed into necrotic symptoms at the tillering stage. Moreover, oswss1 exhibits slightly rolled leaves with irregular epidermal cells, decreased chlorophyll contents, and defective stomata and chloroplasts as compared with the wild type. Map-based cloning revealed that OsWSS1 encodes transmembrane kinase TMK1. Genetic complementary experiments verified that a Leu396Pro amino acid substitution, residing in the highly conserved region of leucine-rich repeat (LRR) domain, was responsible for the phenotypes of oswss1. OsWSS1 was constitutively expressed in all tissues and its encoded protein is localized to the plasma membrane. Mutation of OsWSS1 led to hyper-accumulation of reactive oxygen species (ROS), more severe DNA fragmentation, and cell death than that of the wild-type control. In addition, we found that the expression of senescence-associated genes (SAGs) was significantly higher, while the expression of genes associated with chloroplast development and photosynthesis was significantly downregulated in oswss1 as compared with the wild type. Taken together, our results demonstrated that OsWSS1, a member of TMKs, plays a vital role in the regulation of ROS homeostasis, chloroplast development, and leaf senescence in rice.

Differential gene expression in liver and small intestine from lactating rats compared to age-matched virgin controls detects increased mRNA of cholesterol biosynthetic genes.

BACKGROUND: Lactation increases energy demands four- to five-fold, leading to a two- to three-fold increase in food consumption, requiring a proportional adjustment in the ability of the lactating dam to absorb nutrients and to synthesize critical biomolecules, such as cholesterol, to meet the dietary needs of both the offspring and the dam. The size and hydrophobicity of the bile acid pool increases during lactation, implying an increased absorption and disposition of lipids, sterols, nutrients, and xenobiotics. In order to investigate changes at the transcriptomics level, we utilized an exon array and calculated expression levels to investigate changes in gene expression in the liver, duodenum, jejunum, and ileum of lactating dams when compared against age-matched virgin controls. RESULTS: A two-way mixed models ANOVA was applied to detect differentially expressed genes. Significance calls were defined as a p < 0.05 for the overall physiologic state effect (lactation vs. control), and a within tissue pairwise comparison of p < 0.01. The proportion of false positives, an estimate of the ratio of false positives in the list of differentially expressed genes, was calculated for each tissue. The number of differentially expressed genes was 420 in the liver, 337 in the duodenum, 402 in the jejunum, and 523 in the ileum. The list of differentially expressed genes was in turn analyzed by Ingenuity Pathways Analysis (IPA) to detect biological pathways that were overrepresented. In all tissues, sterol regulatory element binding protein (Srebp)-regulated genes involved in cholesterol synthesis showed increased mRNA expression, with the fewest changes detected in the jejunum. We detected increased Scap mRNA in the liver only, suggesting an explanation for the difference in response to lactation between the liver and small intestine. Expression of Cyp7a1, which catalyzes the rate limiting step in the bile acid biosynthetic pathway, was also significantly increased in liver. In addition, decreased levels of mRNA associated with T-cell signaling were found in the jejunum and ileum. Several members of the Solute Carrier (SLC) and Adenosine Triphosphate Binding Cassette (ABC) superfamilies of membrane transporters were found to be differentially expressed; these genes may play a role in differences in nutrient and xenobiotic absorption and disposition. mRNA expression of SLC39a4_predicted, a zinc transporter, was increased in all tissues, suggesting that it is involved in increased zinc uptake during lactation. Microarray data are available through GEO under GSE19175. CONCLUSIONS: We detected differential expression of mRNA from several pathways in lactating dams, including upregulation of the cholesterol biosynthetic pathway in liver and intestine, consistent with Srebp activation. Differential T-Cell signaling in the two most distal regions of the small intestine (ileum and jejunum) was also noted, as well as differential expression of transporters that likely play a key role in nutrient uptake.

Functional analysis of nonsynonymous single nucleotide polymorphisms of multidrug resistance-associated protein 2 (ABCC2).

BACKGROUND: Multidrug resistance-associated protein 2 (MRP2; ABCC2) mediates the biliary excretion of glutathione, glucuronide, and sulfate conjugates of endobiotics and xenobiotics. Single nucleotide polymorphisms (SNPs) of MRP2 contribute to interindividual variability in drug disposition and ultimately in drug response. OBJECTIVES: To characterize the transport function of human wild-type (WT) MRP2 and four SNP variants, S789F, A1450T, V417I, and T1477M. METHODS: The four SNP variants were expressed in Sf9 cells using recombinant baculovirus infection. The kinetic parameters [Km, (µmol/l); V(max), (pmol/mg/min); the Hill coefficient] of ATP-dependent transport of leukotriene C(4) (LTC(4)), estradiol-3-glucuronide (E(2)3G), estradiol-17ß-glucuronide (E(2)17G), and tauroursodeoxycholic acid (TUDC) were determined in Sf9-derived plasma membrane vesicles. Transport activity was normalized for expression level. RESULTS: The V(max) for transport activity was decreased for all substrates for S789F, and for all substrates except E(2)17G for A1450T. V417I showed decreased apparent affinity for LTC(4), E(2)3G, and E(2)17G, whereas transport was similar between wild-type (WT) and T1477M, except for a modest increase in TUDC transport. Examination of substrate-stimulated MRP2-dependent ATPase activity of S789F and A1450T, SNPs located in MRP2 nucleotide-binding domains (NBDs), demonstrated significantly decreased ATPase activity and only modestly decreased affinity for ATP compared with WT. CONCLUSION: SNPs in the NBDs (S789F in the D-loop of NBD1, or A1450T near the ABC signature motif of NBD2) variably decreased the transport of all substrates. V417I in membrane spanning domain 1 selectively decreased the apparent affinity for the glutathione and glucuronide conjugated substrates, whereas the T1477M SNP in the carboxyl terminus altered only TUDC transport.

Mechanisms for increased expression of cholesterol 7alpha-hydroxylase (Cyp7a1) in lactating rats.

UNLABELLED: Cholesterol 7alpha-hydroxylase (Cyp7a1) and the bile acid pool size are increased 2 to 3-fold in lactating postpartum rats. We investigated the interaction of nuclear receptors with the Cyp7a1 proximal promoter and the expression of regulatory signaling pathways in postpartum rats at day 10 (PPd10) versus female controls to identify the mechanisms of increased expression of Cyp7a1, which is maximal at 16 hours. Liver X receptor (LXRalpha) and RNA polymerase II (RNA Pol II) recruitment to Cyp7a1 chromatin were increased 1.5- and 2.5-fold, respectively, at 16 hours on PPd10. Expression of nuclear receptors farnesoid X receptor (FXR), LXRalpha, liver receptor homolog (LRH-1), hepatocyte nuclear factor 4alpha (HNF4alpha), and short heterodimer partner (SHP) messenger RNA (mRNA) and coactivator peroxisome proliferators-activated receptor gamma coactivator-1alpha (PGC-1alpha) mRNA was unchanged in PPd10 versus controls at 16 hours, whereas chicken ovalbumin upstream transcription factor II (COUP-TFII) was decreased 40% at 16 hours. Investigation of a repressive signaling pathway, the c-Jun-N-terminal kinase (JNK) signaling pathway in PPd10 versus controls, showed decreased mRNA expression of hepatocyte growth factor (HGF; decreased 60% at 16 hours) and tyrosine kinase receptor c-Met (decreased 44%-50% at 16 hours), but these were not accompanied by decreased expression of phosphorylated c-Jun. Importantly, expression of fibroblast growth factor 15 (FGF15) mRNA in the ileum was decreased 70% in PPd10 versus controls, whereas phosphorylated mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (Erk1/2) protein expression in liver was decreased 88% at 16 hours. CONCLUSION: The increased recruitment of LXRalpha, a Cyp7a1 stimulatory pathway, and decreased expression of FGF15 and phosphorylated Erk1/2, a Cyp7a1 repressive pathway, combined to increase Cyp7a1 expression during lactation.

ZmAGA1 Hydrolyzes RFOs Late during the Lag Phase of Seed Germination, Shifting Sugar Metabolism toward Seed Germination Over Seed Aging Tolerance.

Raffinose family oligosaccharides (RFOs) are accumulated during the late stage of seed development and hydrolyzed during seed germination. The process of raffinose hydrolysis during seed germination and how this process affects seed vigor remains unknown. We report here that maize alkaline α-galactosidase 1 (ZmAGA1) protein is translationally induced and is capable of hydrolyzing RFOs as well as a precursor, galactinol, during seed germination. Constitutively overexpressing ZmAGA1 in Arabidopsis decreased both RFOs and galactinol contents of mature, desiccated, and 30 hours after imbibition (HAI) seeds, yet enhanced the seed germination percentage under either salt or somewhat osmotic-stress conditions at earlier times during the time course. However, ZmAGA1 overexpression also decreased the seed aging tolerance of mature, desiccated seeds as compared with wild type (WT) or those overexpressing GFP. Compared to that of WT control seeds, the atsip2 (mutant of Arabidopsis AtSIP2 (seed imbibition protein 2, encoding alkaline α-galactosidase)) seeds have similar RFOs and galactinol contents in mature, desiccated seeds but significantly increased the amount of these metabolites at 30 HAI. This retention of RFOs and galactinol in atsip2 results in seeds that exhibit lowered seed germination percentage under either salt or osmotic stress conditions, and yet, increased seed aging tolerance relative to WT. Similarly, when maize seeds were imbibed in the presence of a specific α-galactosidase inhibitor (1-deoxygalactonojirimycin) as compared to those imbibed in water, greater amounts of raffinose and galactinol were detected; the seeds exhibited decreased seed germination percentages but increased seed aging tolerance. Taken together, these data suggest that both maize seed germination and seed aging tolerance can be simultaneously regulated through careful temporal manipulation of ZmAGA1 expression.

The 5'-untranslated region of multidrug resistance associated protein 2 (MRP2; ABCC2) regulates downstream open reading frame expression through translational regulation.

MRP2 (ABCC2), a member of the ATP binding cassette superfamily of efflux transporters that mediates the apical efflux of organic anions from hepatocytes, enterocytes, and renal epithelial cells, is postulated to undergo post-transcriptional regulation. The MRP2 5'-untranslated region (5'UTR) contains seven upstream start codons and six upstream open reading frames (uORFs). Ribonuclease protection assays in human liver, placenta, kidney, small intestine, and HepG2 cells identified multiple MRP2 transcription initiation sites. We investigated MRP2 5'UTRs [-247 (-247 to -1), -204 (-204 to -1), or -99 (-99 to -1)] for their effects on regulation of gene expression with the use of transient gene expression in HepG2 cells and in vitro translation assays. In HepG2 cells transfected with SV40-MRP2-5'UTR-Luciferase cassettes, luciferase activities of constructs -247 and -204 were significantly lower than that of -99. Disruption of the uORFs at -105 and -74 nucleotides by mutation of ATGs to AAG enhanced luciferase activity significantly without affecting luciferase mRNA expression. The translation efficiencies of T7-5'UTR-Luciferase cassettes determined in vitro were consistent with transfected HepG2 cells and showed that inhibition of translation by the -105 uORF occurred only in the cis configuration and not in the trans configuration and that inhibition of translation by the -105 uORF was independent of the encoded peptide sequence. Characterization of an MRP2 polymorphism, -24C>T, in the MRP2 5'UTR, demonstrated no effect on mRNA expression or downstream ORF translation. These data indicate for the first time that the 5'UTR of MRP2 mRNA transcripts and the uORF at -105 markedly influence MRP2 translation.

Maize VIVIPAROUS1 Interacts with ABA INSENSITIVE5 to Regulate GALACTINOL SYNTHASE2 Expression Controlling Seed Raffinose Accumulation.

Raffinose, an oligosaccharide found in many seeds, plays an important role in seed vigor; however, the regulatory mechanism governing raffinose biosynthesis remains unclear. We report here that maize W22 wild type (WT) seeds, but not W22 viviparous1 ( zmvp1) mutant seeds, start accumulating galactinol and raffinose 28 days after pollination (DAP). Transcriptome analysis of the zmvp1 embryo showed that the expression of GALACTINOL SYNTHASE2 ( GOLS2) was down-regulated relative to WT. Further experiments showed that the expression of ZmGOLS2 was up-regulated by ZmABI5 but not by ZmVP1, and it was further increased by the coexpression of ZmABI5 and ZmVP1 in maize protoplasts. ZmABI5 interacted with ZmVP1, while ZmABI5, but not ZmVP1, directly binds to the ZmGOLS2 promoter. Together, all of the findings suggest that ZmVP1 interacts with ZmABI5 and regulates ZmGOLS2 expression and raffinose accumulation in maize seeds.