Serum dihydroceramides correlate with insulin sensitivity in humans and decrease insulin sensitivity in vitro

Serum ceramides, especially C16:0 and C18:0 species, are linked to CVD risk and insulin resistance, but details of this association are not well understood. We performed this study to quantify a broad range of serum sphingolipids in individuals spanning the physiologic range of insulin sensitivity and to determine if dihydroceramides cause insulin resistance in vitro. As expected, we found that serum triglycerides were significantly greater in individuals with obesity and T2D compared with athletes and lean individuals. Serum ceramides were not significantly different within groups but, using all ceramide data relative to insulin sensitivity as a continuous variable, we observed significant inverse relationships between C18:0, C20:0, and C22:0 species and insulin sensitivity. Interestingly, we found that total serum dihydroceramides and individual species were significantly greater in individuals with obesity and T2D compared with athletes and lean individuals, with C18:0 species showing the strongest inverse relationship to insulin sensitivity. Finally, we administered a physiological mix of dihydroceramides to primary myotubes and found decreased insulin sensitivity in vitro without changing the overall intracellular sphingolipid content, suggesting a direct effect on insulin resistance. These data extend what is known regarding serum sphingolipids and insulin resistance and show the importance of serum dihydroceramides to predict and promote insulin resistance in humans.Supplementary key words: sphingolipids, circulating ceramides, serum, insulin resistance, lipidomics, CVD, T2D, obesity, myotube

Circulating ceramides, especially specific saturated ceramide species, and other sphingolipids are linked to CVD risk and insulin resistance (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). In fact, circulating ceramide and sphingolipid contents predict development of CVD better than some common risk factors such as plasma cholesterol, LDLs, and triglycerides (6, 9, 11, 12). As a result, it was recently proposed that plasma ceramide could be the new cholesterol for assessing risk of CVD (11). Beyond the cross-sectional studies referenced above, there are several lines of evidence supporting the link between ceramides, CVD, and insulin resistance. Plasma ceramide content decreases after insulin-sensitizing gastric bypass surgery and weight loss interventions (13, 14, 15). Animal studies show that ceramides accumulate in atherosclerotic lesions, which may explain the increased risk associated with plasma content (16). However, the relationship of circulating sphingolipids to insulin resistance is not absolute, as insulin-sensitizing treatments do not always change plasma sphingolipid content (17). Combined, most data from epidemiology studies, as well as human interventions and animal models, support the concept that circulating ceramides and sphingolipids are related to insulin resistance and CVD risk.Ceramides circulate primarily bound to lipoproteins and are secreted predominately by the liver. Circulating ceramides are mainly increased in LDL in individuals with obesity (15). Obese rodents have increased hepatic ceramide secretion, which may explain increased plasma ceramide content in individuals with obesity (15). In one mechanistic study, an LDL-ceramide mixture was infused in mice to recapitulate increased plasma ceramide content in obesity, which caused membrane ceramide accumulation, decreased insulin signaling, and a decrease in insulin sensitivity specifically in skeletal muscle, providing evidence for a direct effect of circulating ceramides on tissues (15). Similarly, LDL-ceramide administration to myotubes caused ceramide accumulation, decreased insulin sensitivity, and signaling independent of inflammation. These data indicate that plasma ceramides are not simply markers of insulin resistance but play mechanistic roles in decreasing insulin sensitivity.Ceramides are only one member of the sphingolipid family, and other sphingolipids may also be related to insulin resistance and CVD risk. Lactosylceramides and glucosylceramides are sphingolipids that also accumulate in atherosclerotic plaques and therefore may be involved in the CVD process (18). Sphingomyelins are the most abundant sphingolipids circulating in lipoproteins and, while they are positively related to obesity and waist circumference, they are not correlated to insulin sensitivity in cross-sectional human studies (5, 19). Dihydroceramides are immediate precursors to ceramide synthesis and are negatively related to insulin sensitivity (20, 21) and insulin secretion (21), are positively related to waist circumference (22), are elevated in plasma of individuals with prediabetes and T2D compared with controls (23), and predict development of diabetes 9 years before onset (21). Despite strong evidence linking plasma dihydroceramides to decreased insulin sensitivity, mechanistic studies to determine if circulating dihydroceramides cause insulin resistance are lacking.To address this knowledge gap, we performed the current study to assess serum sphingolipids in humans across the metabolic spectrum as well as determine if dihydroceramides induce insulin resistance in vitro.     

The small GTPase RAB10 regulates endosomal recycling of the LDL receptor and transferrin receptor in hepatocytes

The low-density lipoprotein receptor (LDLR) mediates the hepatic uptake of circulating low-density lipoproteins (LDLs), a process that modulates the development of atherosclerotic cardiovascular disease. We recently identified RAB10, encoding a small GTPase, as a positive regulator of LDL uptake in hepatocellular carcinoma cells (HuH7) in a genome-wide CRISPR screen, though the underlying molecular mechanism for this effect was unknown. We now report that RAB10 regulates hepatocyte LDL uptake by promoting the recycling of endocytosed LDLR from RAB11-positive endosomes to the plasma membrane. We also show that RAB10 similarly promotes the recycling of the transferrin receptor, which binds the transferrin protein that mediates the transport of iron in the blood, albeit from a distinct RAB4-positive compartment. Taken together, our findings suggest a model in which RAB10 regulates LDL and transferrin uptake by promoting both slow and rapid recycling routes for their respective receptor proteins.Supplementary key words: low density lipoprotein receptor, receptors, protein trafficking, cholesterol, lipoproteins, CRISPR screen, HuH7 cells, endocytosis, RAB10, RAB11

An elevated level of circulating low-density lipoprotein (LDL) cholesterol is a major risk factor for atherosclerotic cardiovascular diseases, including myocardial infarction and stroke (1, 2, 3, 4, 5, 6, 7). Regulation of plasma cholesterol is governed by a complex interplay between dietary absorption, de novo biosynthesis, and clearance from the bloodstream. Therapeutic targeting of LDL clearance has been a highly successful strategy for the prevention and treatment of atherosclerosis. LDL clearance is mediated by the LDL receptor (LDLR), a cell-surface glycoprotein that directly binds to the apolipoprotein B component of LDL particles and triggers clathrin-mediated endocytosis. The acidic environment of the endosomal lumen induces complex dissociation, with LDL subsequently transported to the lysosome for hydrolysis, and free LDLR recycled back to the plasma membrane (8, 9). Many regulatory proteins affecting the endocytic pathway and cell-surface expression of LDLR have been identified, including PCSK9, a negative regulator that redirects LDLR to the lysosome for degradation (10), and IDOL, a ubiquitin ligase that induces proteasomal degradation of LDLR (11, 12). Although much is known about the regulation of LDLR expression and endocytosis, questions remain concerning the molecular determinants of LDLR recycling.We recently reported a genome-wide CRISPR screen for modifiers of LDL uptake in HuH7 cells (13). This screen identified RAB10, a small GTPase known to mediate trafficking of vesicles between intracellular compartments, as a key regulator of LDL uptake. Deletion of RAB10 decreased cellular endocytosis of LDL but increased accumulation of another endocytic cargo, transferrin. The receptors for LDL (LDLR) and transferrin receptor (TFR) are both endocytosed from the cell surface via clathrin-coated vesicles and transported through intracellular recycling pathways (14, 15, 16, 17, 18, 19, 20). In this study, we investigated the role of RAB10 in LDL and transferrin endocytosis. Our results demonstrate that GTP-bound RAB10 positively regulates the activity of LDLR and TFR by accelerating the recycling of both proteins to the plasma membrane.     

The nuclease activity of DNA2 promotes exonuclease 1–independent mismatch repair

The DNA mismatch repair (MMR) system is a major DNA repair system that corrects DNA replication errors. In eukaryotes, the MMR system functions via mechanisms both dependent on and independent of exonuclease 1 (EXO1), an enzyme that has multiple roles in DNA metabolism. Although the mechanism of EXO1-dependent MMR is well understood, less is known about EXO1-independent MMR. Here, we provide genetic and biochemical evidence that the DNA2 nuclease/helicase has a role in EXO1-independent MMR. Biochemical reactions reconstituted with purified human proteins demonstrated that the nuclease activity of DNA2 promotes an EXO1-independent MMR reaction via a mismatch excision-independent mechanism that involves DNA polymerase δ. We show that DNA polymerase ε is not able to replace DNA polymerase δ in the DNA2-promoted MMR reaction. Unlike its nuclease activity, the helicase activity of DNA2 is dispensable for the ability of the protein to enhance the MMR reaction. Further examination established that DNA2 acts in the EXO1-independent MMR reaction by increasing the strand-displacement activity of DNA polymerase δ. These data reveal a mechanism for EXO1-independent mismatch repair.

The mismatch repair (MMR) system has been conserved from bacteria to humans (1, 2). It promotes genome stability by suppressing spontaneous and DNA damage-induced mutations (1, 3, 4, 5, 6, 7, 8, 9, 10, 11). The key function of the MMR system is the correction of DNA replication errors that escape the proofreading activities of replicative DNA polymerases (1, 4, 5, 6, 7, 8, 9, 10, 12). In addition, the MMR system removes mismatches formed during strand exchange in homologous recombination, suppresses homeologous recombination, initiates apoptosis in response to irreparable DNA damage caused by several anticancer drugs, and contributes to instability of triplet repeats and alternative DNA structures (1, 4, 5, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18). The principal components of the eukaryotic MMR system are MutSα (MSH2-MSH6 heterodimer), MutLα (MLH1-PMS2 heterodimer in humans and Mlh1-Pms1 heterodimer in yeast), MutSβ (MSH2-MSH3 heterodimer), proliferating cell nuclear antigen (PCNA), replication factor C (RFC), exonuclease 1 (EXO1), RPA, and DNA polymerase δ (Pol δ). Loss-of-function mutations in the MSH2, MLH1, MSH6, and PMS2 genes of the human MMR system cause Lynch and Turcot syndromes, and hypermethylation of the MLH1 promoter is responsible for ∼15% of sporadic cancers in several organs (19, 20). MMR deficiency leads to cancer initiation and progression via a multistage process that involves the inactivation of tumor suppressor genes and action of oncogenes (21).MMR occurs behind the replication fork (22, 23) and is a major determinant of the replication fidelity (24). The correction of DNA replication errors by the MMR system increases the replication fidelity by ∼100 fold (25). Strand breaks in leading and lagging strands as well as ribonucleotides in leading strands serve as signals that direct the eukaryotic MMR system to remove DNA replication errors (26, 27, 28, 29, 30). MMR is more efficient on the lagging than the leading strand (31). The substrates for MMR are all six base–base mismatches and 1 to 13-nt insertion/deletion loops (25, 32, 33, 34). Eukaryotic MMR commences with recognition of the mismatch by MutSα or MutSβ (32, 34, 35, 36). MutSα is the primary mismatch-recognition factor that recognizes both base–base mismatches and small insertion/deletion loops whereas MutSβ recognizes small insertion/deletion loops (32, 34, 35, 36, 37). After recognizing the mismatch, MutSα or MutSβ cooperates with RFC-loaded PCNA to activate MutLα endonuclease (38, 39, 40, 41, 42, 43). The activated MutLα endonuclease incises the discontinuous daughter strand 5′ and 3′ to the mismatch. A 5'' strand break formed by MutLα endonuclease is utilized by EXO1 to enter the DNA and excise a discontinuous strand portion encompassing the mismatch in a 5''→3′ excision reaction stimulated by MutSα/MutSβ (38, 44, 45). The generated gap is filled in by the Pol δ holoenzyme, and the nick is ligated by a DNA ligase (44, 46, 47). DNA polymerase ε (Pol ε) can substitute for Pol δ in the EXO1-dependent MMR reaction, but its activity in this reaction is much lower than that of Pol δ (48). Although MutLα endonuclease is essential for MMR in vivo, 5′ nick-dependent MMR reactions reconstituted in the presence of EXO1 are MutLα-independent (44, 47, 49).EXO1 deficiency in humans does not seem to cause significant cancer predisposition (19). Nevertheless, it is known that Exo1^-/- mice are susceptible to the development of lymphomas (50). Genetic studies in yeast and mice demonstrated that EXO1 inactivation causes only a modest defect in MMR (50, 51, 52, 53). In agreement with these genetic studies, a defined human EXO1-independent MMR reaction that depends on the strand-displacement DNA synthesis activity of Pol δ holoenzyme to remove the mismatch was reconstituted (54). Furthermore, an EXO1-independent MMR reaction that occurred in a mammalian cell extract system without the formation of a gapped excision intermediate was observed (54). Together, these findings implicated the strand-displacement activity of Pol δ holoenzyme in EXO1-independent MMR.In this study, we investigated DNA2 in the context of MMR. DNA2 is an essential multifunctional protein that has nuclease, ATPase, and 5''→3′ helicase activities (55, 56, 57). Previous research ascertained that DNA2 removes long flaps during Okazaki fragment maturation (58, 59, 60), participates in the resection step of double-strand break repair (61, 62, 63), initiates the replication checkpoint (64), and suppresses the expansions of GAA repeats (65). We have found in vivo and in vitro evidence that DNA2 promotes EXO1-independent MMR. Our data have indicated that the nuclease activity of DNA2 enhances the strand-displacement activity of Pol δ holoenzyme in an EXO1-independent MMR reaction.     

Concerted actions of DnaA complexes with DNA-unwinding sequences within and flanking replication origin oriC promote DnaB helicase loading

Unwinding of the replication origin and loading of DNA helicases underlie the initiation of chromosomal replication. In Escherichia coli, the minimal origin oriC contains a duplex unwinding element (DUE) region and three (Left, Middle, and Right) regions that bind the initiator protein DnaA. The Left/Right regions bear a set of DnaA-binding sequences, constituting the Left/Right-DnaA subcomplexes, while the Middle region has a single DnaA-binding site, which stimulates formation of the Left/Right-DnaA subcomplexes. In addition, a DUE-flanking AT-cluster element (TATTAAAAAGAA) is located just outside of the minimal oriC region. The Left-DnaA subcomplex promotes unwinding of the flanking DUE exposing TT[A/G]T(T) sequences that then bind to the Left-DnaA subcomplex, stabilizing the unwound state required for DnaB helicase loading. However, the role of the Right-DnaA subcomplex is largely unclear. Here, we show that DUE unwinding by both the Left/Right-DnaA subcomplexes, but not the Left-DnaA subcomplex only, was stimulated by a DUE-terminal subregion flanking the AT-cluster. Consistently, we found the Right-DnaA subcomplex–bound single-stranded DUE and AT-cluster regions. In addition, the Left/Right-DnaA subcomplexes bound DnaB helicase independently. For only the Left-DnaA subcomplex, we show the AT-cluster was crucial for DnaB loading. The role of unwound DNA binding of the Right-DnaA subcomplex was further supported by in vivo data. Taken together, we propose a model in which the Right-DnaA subcomplex dynamically interacts with the unwound DUE, assisting in DUE unwinding and efficient loading of DnaB helicases, while in the absence of the Right-DnaA subcomplex, the AT-cluster assists in those processes, supporting robustness of replication initiation.

The initiation of bacterial DNA replication requires local duplex unwinding of the chromosomal replication origin oriC, which is regulated by highly ordered initiation complexes. In Escherichia coli, the initiation complex contains oriC, the ATP-bound form of the DnaA initiator protein (ATP–DnaA), and the DNA-bending protein IHF (Fig. 1, A and B), which promotes local unwinding of oriC (1, 2, 3, 4). Upon this oriC unwinding, two hexamers of DnaB helicases are bidirectionally loaded onto the resultant single-stranded (ss) region with the help of the DnaC helicase loader (Fig. 1B), leading to bidirectional chromosomal replication (5, 6, 7, 8). However, the fundamental mechanism underlying oriC-dependent bidirectional DnaB loading remains elusive.Open in a separate windowFigure 1Schematic structures of oriC, DnaA, and the initiation complexes. A, the overall structure of oriC. The minimal oriC region and the AT-cluster region are indicated. The sequence of the AT-cluster−DUE (duplex-unwinding element) region is also shown below. The DUE region (DUE; pale orange bars) contains three 13-mer repeats: L-DUE, M-DUE, and R-DUE. DnaA-binding motifs in M/R-DUE, TT(A/G)T(T), are indicated by red characters. The AT-cluster region (AT cluster; brown bars) is flanked by DUE outside of the minimal oriC. The DnaA-oligomerization region (DOR) consists of three subregions called Left-, Middle-, and Right-DOR. B, model for replication initiation. DnaA is shown as light brown (for domain I–III) and darkbrown (for domain IV) polygons (right panel). ATP–DnaA forms head-to-tail oligomers on the Left- and Right-DORs (left panel). The Middle-DOR (R2 box)-bound DnaA interacts with DnaA bound to the Left/Right-DORs using domain I, but not domain III, stimulating DnaA assembly. IHF, shown as purple hexagons, bends DNA >160° and supports DUE unwinding by the DnaA complexes. M/R-DUE regions are efficiently unwound. Unwound DUE is recruited to the Left-DnaA subcomplex and mainly binds to R1/R5M-bound DnaA molecules. The sites of ssDUE-binding B/H-motifs V211 and R245 of R1/R5M-bound DnaA molecules are indicated (pink). Two DnaB homohexamer helicases (light green) are recruited and loaded onto the ssDUE regions with the help of the DnaC helicase loader (cyan). ss, single stranded.The minimal oriC region consists of the duplex unwinding element (DUE) and the DnaA oligomerization region (DOR), which contains specific arrays of 9-mer DnaA-binding sites (DnaA boxes) with the consensus sequence TTA[T/A]NCACA (Fig. 1A) (3, 4). The DUE underlies the local unwinding and contains 13-mer AT-rich sequence repeats named L-, M-, and R-DUE (9). The M/R-DUE region includes TT[A/G]T(A) sequences with specific affinity for DnaA (10). In addition, a DUE-flanking AT-cluster (TATTAAAAAGAA) region resides just outside of the minimal oriC (Fig. 1A) (11). The DOR is divided into three subregions, the Left-, Middle-, and Right-DORs, where DnaA forms structurally distinct subcomplexes (Fig. 1A) (8, 12, 13, 14, 15, 16, 17). The Left-DOR contains high-affinity DnaA box R1, low-affinity boxes R5M, τ1−2, and I1-2, and an IHF-binding region (17, 18, 19, 20). The τ1 and IHF-binding regions partly overlap (17).In the presence of IHF, ATP–DnaA molecules cooperatively bind to R1, R5M, τ2, and I1-2 boxes in the Left-DOR, generating the Left-DnaA subcomplex (Fig. 1B) (8, 17). Along with IHF causing sharp DNA bending, the Left-DnaA subcomplex plays a leading role in DUE unwinding and subsequent DnaB loading. The Middle-DOR contains moderate-affinity DnaA box R2. Binding of DnaA to this box stimulates DnaA assembly in the Left- and Right-DORs using interaction by DnaA N-terminal domain (Fig. 1B; also see below) (8, 12, 14, 16, 21). The Right-DOR contains five boxes (C3-R4 boxes) and cooperative binding of ATP–DnaA molecules to these generates the Right-DnaA subcomplex (Fig. 1B) (12, 18). This subcomplex is not essential for DUE unwinding and plays a supportive role in DnaB loading (8, 15, 17). The Left-DnaA subcomplex interacts with DnaB helicase, and the Right-DnaA subcomplex has been suggested to play a similar role (Fig. 1B) (8, 13, 16).In the presence of ATP–DnaA, M- and R-DUE adjacent to the Left-DOR are predominant sites for in vitro DUE unwinding: unwinding of L-DUE is less efficient than unwinding of the other two (Fig. 1B) (9, 22, 23). Deletion of L-DUE or the whole DUE inhibits replication of oriC in vitro moderately or completely, respectively (23). A chromosomal oriC Δ(AT-cluster−L-DUE) mutant with an intact DOR, as well as deletion of Right-DOR, exhibits limited inhibition of replication initiation, whereas the synthetic mutant combining the two deletions exhibits severe inhibition of cell growth (24). These studies suggest that AT-cluster−L-DUE regions stimulate replication initiation in a manner concerted with Right-DOR, although the underlying mechanisms remain elusive.DnaA consists of four functional domains (Fig. 1B) (4, 25). Domain I supports weak domain I–domain I interaction and serves as a hub for interaction with various proteins such as DnaB helicase and DiaA, which stimulates ATP–DnaA assembly at oriC (26, 27, 28, 29, 30). Two or three domain I molecules of the oriC–DnaA subcomplex bind a single DnaB hexamer, forming a stable higher-order complex (7). Domain II is a flexible linker (28, 31). Domain III contains AAA+ (ATPase associated with various cellular activities) motifs essential for ATP/ADP binding, ATP hydrolysis, and DnaA–DnaA interactions in addition to specific sites for ssDUE binding and a second, weak interaction with DnaB helicase (1, 4, 8, 10, 19, 25, 32, 33, 34, 35). Domain IV bears a helix-turn-helix motif with specific affinity for the DnaA box (36).As in typical AAA+ proteins, a head-to-tail interaction underlies formation of ATP–DnaA pentamers on the DOR, where the AAA+ arginine-finger motif Arg285 recognizes ATP bound to the adjacent DnaA protomer, promoting cooperative ATP–DnaA binding (Fig. 1B) (19, 32). DnaA ssDUE-binding H/B-motifs (Val211 and Arg245) in domain III sustain stable unwinding by directly binding to the T-rich (upper) strand sequences TT[A/G]T(A) within the unwound M/R-DUE (Fig. 1B) (8, 10). Val211 residue is included in the initiator-specific motif of the AAA+ protein family (10). For DUE unwinding, ssDUE is recruited to the Left-DnaA subcomplex via DNA bending by IHF and directly interacts with H/B-motifs of DnaA assembled on Left-DOR, resulting in stable DUE unwinding competent for DnaB helicase loading; in particular, DnaA protomers bound to R1 and R5M boxes play a crucial role in the interaction with M/R-ssDUE (Fig. 1B) (8, 10, 17). Collectively, these mechanisms are termed ssDUE recruitment (4, 17, 37).Two DnaB helicases are thought to be loaded onto the upper and lower strands of the region including the AT-cluster and DUE, with the aid of interactions with DnaC and DnaA (Fig. 1B) (25, 38, 39). DnaC binding modulates the closed ring structure of DnaB hexamer into an open spiral form for entry of ssDNA (40, 41, 42, 43). Upon ssDUE loading of DnaB, DnaC is released from DnaB in a manner stimulated by interactions with ssDNA and DnaG primase (44, 45). Also, the Left- and Right-DnaA subcomplexes, which are oriented opposite to each other, could regulate bidirectional loading of DnaB helicases onto the ssDUE (Fig. 1B) (7, 8, 35). Similarly, recent works suggest that the origin complex structure is bidirectionally organized in both archaea and eukaryotes (1, 46). In Saccharomyces cerevisiae, two origin recognition complexes containing AAA+ proteins bind to the replication origin region in opposite orientations; this, in turn, results in efficient loading of two replicative helicases, leading to head-to-head interactions in vitro (46). Consistent with this, origin recognition complex dimerization occurs in the origin region during the late M-G1 phase (47). The fundamental mechanism of bidirectional origin complexes might be widely conserved among species.In this study, we analyzed various mutants of oriC and DnaA in reconstituted systems to reveal the regulatory mechanisms underlying DUE unwinding and DnaB loading. The Right-DnaA subcomplex assisted in the unwinding of oriC, dependent upon an interaction with L-DUE, which is important for efficient loading of DnaB helicases. The AT-cluster region adjacent to the DUE promoted loading of DnaB helicase in the absence of the Right-DnaA subcomplex. Consistently, the ssDNA-binding activity of the Right-DnaA subcomplex sustained timely initiation of growing cells. These results indicate that DUE unwinding and efficient loading of DnaB helicases are sustained by concerted actions of the Left- and Right-DnaA subcomplexes. In addition, loading of DnaB helicases are sustained by multiple mechanisms that ensure robust replication initiation, although the complete mechanisms are required for precise timing of initiation during the cell cycle.     

Nitrogen Use Efficiency Is Mediated by Vacuolar Nitrate Sequestration Capacity in Roots of Brassica napus

Enhancing nitrogen use efficiency (NUE) in crop plants is an important breeding target to reduce excessive use of chemical fertilizers, with substantial benefits to farmers and the environment. In Arabidopsis (Arabidopsis thaliana), allocation of more NO₃⁻ to shoots was associated with higher NUE; however, the commonality of this process across plant species have not been sufficiently studied. Two Brassica napus genotypes were identified with high and low NUE. We found that activities of V-ATPase and V-PPase, the two tonoplast proton-pumps, were significantly lower in roots of the high-NUE genotype (Xiangyou15) than in the low-NUE genotype (814); and consequently, less vacuolar NO₃⁻ was retained in roots of Xiangyou15. Moreover, NO₃⁻ concentration in xylem sap, [¹⁵N] shoot:root (S:R) and [NO₃⁻] S:R ratios were significantly higher in Xiangyou15. BnNRT1.5 expression was higher in roots of Xiangyou15 compared with 814, while BnNRT1.8 expression was lower. In both B. napus treated with proton pump inhibitors or Arabidopsis mutants impaired in proton pump activity, vacuolar sequestration capacity (VSC) of NO₃⁻ in roots substantially decreased. Expression of NRT1.5 was up-regulated, but NRT1.8 was down-regulated, driving greater NO₃⁻ long-distance transport from roots to shoots. NUE in Arabidopsis mutants impaired in proton pumps was also significantly higher than in the wild type col-0. Taken together, these data suggest that decrease in VSC of NO₃⁻ in roots will enhance transport to shoot and essentially contribute to higher NUE by promoting NO₃⁻ allocation to aerial parts, likely through coordinated regulation of NRT1.5 and NRT1.8.China is the largest consumer of nitrogen (N) fertilizer in the world; however, the average N use efficiency (NUE) in fertilizer is only around 35%, suggesting considerable potential for improvements (Shen et al., 2003; Wang et al., 2014). With the high amounts of N-fertilizer being used, crop yields are declining in some areas, where application is exceeding the optimum required for local field crops (Shen et al., 2003; Miller and Smith, 2008; Xu et al., 2012). The extremely low NUE results in waste of resources and environmental contamination, and also presents serious hazards for human health (Xu et al., 2012; Chen et al., 2014). Consequently, exploiting the maximum potential for improving NUE in crop plants will have practical significance for agriculture production and the environment (Zhang et al., 2010; Schroeder et al., 2013; Wang et al., 2014). Elucidating the genetic and physiological regulatory mechanisms governing NUE in plants will allow breeding crops and varieties with higher NUE.Ammonium (NH₄⁺) and nitrate (NO₃⁻) are the main N species absorbed and utilized by crops, and NO₃⁻ accumulation and utilization are of major emphasis for N nutrient studies in dry land crops, such as Brassica napus. Several studies revealed the close relationship between NO₃⁻ content and NUE in plant tissues (Shen et al., 2003; Zhang et al., 2012; Tang et al., 2013; Han et al., 2015a). When plants are sufficiently illuminated, NO₃⁻ assimilation efficiency significantly increase in shoots compared with roots (Smirnoff and Stewart, 1985; Tang et al., 2013). Consequently, under daytime with optimal illumination, higher proportion of NO₃⁻ in plant tissue is transported from root to shoot, as an advantageous physiological adaptation that reduces the cost of energy for metabolism (Tang et al., 2013). NO₃⁻ assimilation in plant shoots can therefore take advantage of solar energy while improving NUE (Smirnoff and Stewart, 1985; Andrews, 1986; Tang et al., 2012, 2013).The NO₃⁻ long-distance transport and distribution between root and shoot is regulated by two genes encoding long transport mechanisms. NRT1.5 is responsible for xylem NO₃⁻ loading, while NRT1.8 is responsible for xylem NO₃⁻ unloading (Lin et al., 2008; Li et al., 2010). Expression of the two genes is influenced by NO₃⁻ concentration. NRT1.5 is strongly induced by NO₃⁻ (Lin et al., 2008), while NRT1.8 expression is extremely up-regulated in nrt1.5 mutants (Chen et al., 2012). A negative correlation between the extents of expression of the two genes was observed when plants are subjected to abiotic stresses (Chen et al., 2012). Moreover, expression of NRT1.5 is strongly inhibited by 1-aminocyclopropane-1-carboxylic acid (ACC) and methyl jasmonate (MeJA), whereas the expression of NRT1.8 is significantly up-regulated (Zhang et al., 2014). Based on these studies, we argue that the expression and functioning of NO₃⁻ long-distance transport genes NRT1.5 and NRT1.8 are regulated by cytosolic NO₃⁻ concentration. In addition, the vacuolar and cytosolic NO₃⁻ distribution is likely regulated by proton pumps located within the tonoplast (V-ATPase and V-PPase; Granstedt and Huffaker, 1982; Glass et al., 2002; Krebs et al., 2010). Therefore, NO₃⁻ use efficiency must be affected by NO₃⁻ long-distant transport (between shoot and root) and short-distant transport (between vacuole and cytosol). However, the physiological mechanisms controlling this regulation are still obscure.Previous studies showed that the chloride channel protein (CLCa) is mainly responsible for vacuole NO₃⁻ short-distance transport, as it is the main channel for NO₃⁻ movement between the vacuoles and cytosol (De Angeli et al., 2006; Wege et al., 2014). The vacuole proton-pumps (V-ATPase and V-PPase) located in the tonoplast supply energy for active transport of NO₃⁻ and accumulation within the vacuole (Gaxiola et al., 2001; Brüx et al., 2008; Krebs et al., 2010). Despite the fact about 90% of the volume of mature plant cells is occupied by vacuoles, vacuolar NO₃⁻ cannot be efficiently assimilated because the enzyme nitrate reductase (NR) is cytosolic (Shen et al., 2003; Han et al., 2015a). However, retranslocation of NO₃⁻ from the vacuole to the cytosol will permit its immediate assimilation and utilization.Generally, NO₃⁻ concentrations in plant cell vacuoles and the cytoplasm are in the range of 30–50 mol m⁻³ and 3–5 mol m⁻³, respectively (Martinoia et al., 1981, 2000). Because vacuoles are obviously the organelle for high NO₃⁻ accumulation and storage in plant tissues, their function in NO₃⁻ use efficiency cannot be ignored (Martinoia et al., 1981; Zhang et al., 2012; Han et al., 2015b). NO₃⁻ assimilatory system in the cytoplasm is sufficient for its assimilation when it is transported out of the vacuoles. Therefore, NO₃⁻ use efficiency could in part be dependent on vacuolar-cytosolic NO₃⁻ short-distance transport in plant tissues (Martinoia et al., 1981; Shen et al., 2003; Zhang et al., 2012; Han et al., 2015a).Evidently, NO₃⁻ use efficiency is regulated by both NO₃⁻ long-distance transport from root to shoot and short-distance transport and distribution between vacuoles and cytoplasm within cells (Glass et al., 2002; Dechorgnat et al., 2011; Han et al., 2015a). Although vacuoles compartment excess NO₃⁻ that accumulates in plant cells (Granstedt and Huffaker, 1982; Krebs et al., 2010), neither NO₃⁻ inducible NR genes (NIA1 and NIA2; Fan et al., 2007; Han et al., 2015a) nor the NO₃⁻ long-distance transport gene NRT1.5 (Lin et al., 2008) are regulated by vacuolar NO₃⁻, even though they are essential for NO₃⁻ assimilation. Only NO₃⁻ transported from the vacuole to the cytosol can play a role in regulating NO₃⁻ inducible genes. Consequently, we argue that both NO₃⁻ assimilation in cells and its long-distance transport from root to shoot are regulated by cytosolic NO₃⁻ concentration. However, this hypothesis needs to be substantiated. The mechanisms underlying both NO₃⁻ short-distance (Gaxiola et al., 2001; De Angeli et al., 2006; Brüx et al., 2008; Krebs et al., 2010) and long-distance transport (Lin et al., 2008; Li et al., 2010) have been previously investigated, yet the underlying mechanisms regulating the flux of NO₃⁻ and the obvious relationship between the two transport pathways, as well as their relation to NUE, are not well understood.The NRT family of genes play a partial role in vacuolar NO₃⁻ accumulation in petioles (Chiu et al., 2004) and seed tissues (Chopin et al., 2007), whereas the proton pumps and CLCa system in the tonoplast play a major role in accumulating NO₃⁻ in vacuoles (Gaxiola et al., 2001; De Angeli et al., 2006; Brüx et al., 2008; Krebs et al., 2010). The vacuolar NO₃⁻ short-distance transport system is spread throughout the plant tissues and is the principal means by which vacuolar NO₃⁻ short-distance transport and distribution is controlled (De Angeli et al., 2006; Krebs et al., 2010).The NRT genes seem to work synergistically to control NO₃⁻ long-distance transport between roots and shoots. NRT1.9 is responsible for NO₃⁻ loading into the phloem (Wang and Tsay, 2011), whereas NO₃⁻ loading and unloading into xylem are regulated by NRT1.5 and NRT1.8, respectively (Lin et al., 2008; Li et al.; 2010). Phloem transport mainly involves organic N; the inorganic-N (NO₃⁻) concentrations in the phloem sap are typically very low, ranging from one-tenth to one-hundredth of that of the inorganic-N in xylem sap (Lin et al., 2008; Fan et al., 2009). Therefore, this study focused on NO₃⁻ short-distance transport mediated through the tonoplast proton pumps and the CLCa system and the long-distant transport mechanisms responsible for xylem NO₃⁻ loading and unloading via NRT1.5 and NRT1.8, respectively.Questions related to how long- and short-distance transport of NO₃⁻ are coupled in plant tissues and their role in determining NUE were addressed using a pair of high- and low-NUE B. napus genotypes and Arabidopsis (Arabidopsis thaliana). Application of proton pump inhibitors and ACC in the former, and use of mutants with defective proton pumps in the latter, allowed experimental distinction of the physiological mechanisms regulating these processes. Data presented here provide strong evidence from both model plants supporting this linkage and strongly suggest that cytosolic NO₃⁻ concentration in roots regulates NO₃⁻ long-distance transport from roots to shoots. We also investigated how NO₃⁻ concentration in plant tissues would be affected by NO₃⁻ long-distance transport, vacuolar NO₃⁻ sequestration, and the ensuing relationship with NO₃⁻ use efficiency. We also proposed the physiological mechanisms likely to be important for enhancing NO₃⁻ use efficiency in plants. These findings will provide scientific rationales for improving NUE in important industrial and food crops.     

Responses of Arabidopsis and Wheat to Rising CO2 Depend on Nitrogen Source and Nighttime CO2 Levels

A major contributor to the global carbon cycle is plant respiration. Elevated atmospheric CO₂ concentrations may either accelerate or decelerate plant respiration for reasons that have been uncertain. We recently established that elevated CO₂ during the daytime decreases plant mitochondrial respiration in the light and protein concentration because CO₂ slows the daytime conversion of nitrate (NO₃−) into protein. This derives in part from the inhibitory effect of CO₂ on photorespiration and the dependence of shoot NO₃− assimilation on photorespiration. Elevated CO₂ also inhibits the translocation of nitrite into the chloroplast, a response that influences shoot NO₃− assimilation during both day and night. Here, we exposed Arabidopsis (Arabidopsis thaliana) and wheat (Triticum aestivum) plants to daytime or nighttime elevated CO₂ and supplied them with NO₃− or ammonium as a sole nitrogen (N) source. Six independent measures (plant biomass, shoot NO₃−, shoot organic N, ¹⁵N isotope fractionation, ¹⁵NO₃⁻ assimilation, and the ratio of shoot CO₂ evolution to O₂ consumption) indicated that elevated CO₂ at night slowed NO₃− assimilation and thus decreased dark respiration in the plants reliant on NO₃−. These results provide a straightforward explanation for the diverse responses of plants to elevated CO₂ at night and suggest that soil N source will have an increasing influence on the capacity of plants to mitigate human greenhouse gas emissions.The CO₂ concentration in Earth’s atmosphere has increased from about 270 to 400 µmol mol^–1 since 1800, and may double before the end of the century (Intergovernmental Panel on Climate Change, 2013). Plant responses to such increases are highly variable, but plant nitrogen (N) concentrations generally decline under elevated CO₂ (Cotrufo et al., 1998; Long et al., 2004). One explanation for this decline is that CO₂ inhibits nitrate (NO₃−) assimilation into protein in the shoots of C₃ plants during the daytime (Bloom et al., 2002, 2010, 2012, 2014; Cheng et al., 2012; Pleijel and Uddling, 2012; Myers et al., 2014; Easlon et al., 2015; Pleijel and Högy, 2015). This derives in part from the inhibitory effect of CO₂ on photorespiration (Foyer et al., 2009) and the dependence of shoot NO₃− assimilation on photorespiration (Rachmilevitch et al., 2004; Bloom, 2015).A key factor in global carbon budgets is plant respiration at night (Amthor, 1991; Farrar and Williams, 1991; Drake et al., 1999; Leakey et al., 2009). Nighttime elevated CO₂ may inhibit, have a negligible effect on, or stimulate dark respiration, depending on the plant species (Bunce, 2001, 2003; Wang and Curtis, 2002), plant development stage (Wang et al., 2001; Li et al., 2013), experimental approach (Griffin et al., 1999; Baker et al., 2000; Hamilton et al., 2001; Bruhn et al., 2002; Jahnke and Krewitt, 2002; Bunce, 2004), and total N supply (Markelz et al., 2014). The current study is, to our knowledge, the first to examine the influence of N source, NO₃− versus ammonium (NH₄+), on plant dark respiration at elevated CO₂ during the night.Plant organic N compounds account for less than 5% of the total dry weight of a plant, but conversion of NO₃− into organic N expends about 25% of the total energy in shoots (Bloom et al., 1989) and roots (Bloom et al., 1992). During the day, photorespiration supplies a portion of the energy (Rachmilevitch et al., 2004; Foyer et al., 2009), but at night, this energetic cost is borne entirely by the respiration of C substrates (Amthor, 1995) and may divert a substantial amount of reductant from the mitochondrial electron transport chain (Cousins and Bloom, 2004). The relative importance of NO₃− assimilation at night versus the day, however, is still a matter of intense debate (Nunes-Nesi et al., 2010). Here, we estimated NO₃− assimilation using several independent methods and show in Arabidopsis (Arabidopsis thaliana) and wheat (Triticum aestivum), two diverse C₃ plants, that NO₃− assimilation at night can be substantial, and that elevated CO₂ at night inhibits this process.     

Focus on Ethylene: Ethylene Biosynthesis Is Promoted by Very-Long-Chain Fatty Acids during Lysigenous Aerenchyma Formation in Rice Roots

In rice (Oryza sativa) roots, lysigenous aerenchyma, which is created by programmed cell death and lysis of cortical cells, is constitutively formed under aerobic conditions, and its formation is further induced under oxygen-deficient conditions. Ethylene is involved in the induction of aerenchyma formation. reduced culm number1 (rcn1) is a rice mutant in which the gene encoding the ATP-binding cassette transporter RCN1/OsABCG5 is defective. Here, we report that the induction of aerenchyma formation was reduced in roots of rcn1 grown in stagnant deoxygenated nutrient solution (i.e. under stagnant conditions, which mimic oxygen-deficient conditions in waterlogged soils). 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a key enzyme in ethylene biosynthesis. Stagnant conditions hardly induced the expression of ACS1 in rcn1 roots, resulting in low ethylene production in the roots. Accumulation of saturated very-long-chain fatty acids (VLCFAs) of 24, 26, and 28 carbons was reduced in rcn1 roots. Exogenously supplied VLCFA (26 carbons) increased the expression level of ACS1 and induced aerenchyma formation in rcn1 roots. Moreover, in rice lines in which the gene encoding a fatty acid elongase, CUT1-LIKE (CUT1L; a homolog of the gene encoding Arabidopsis CUT1, which is required for cuticular wax production), was silenced, both ACS1 expression and aerenchyma formation were reduced. Interestingly, the expression of ACS1, CUT1L, and RCN1/OsABCG5 was induced predominantly in the outer part of roots under stagnant conditions. These results suggest that, in rice under oxygen-deficient conditions, VLCFAs increase ethylene production by promoting 1-aminocyclopropane-1-carboxylic acid biosynthesis in the outer part of roots, which, in turn, induces aerenchyma formation in the root cortex.Aerenchyma formation is a morphological adaptation of plants to complete submergence and waterlogging of the soil, and facilitates internal gas diffusion (Armstrong, 1979; Jackson and Armstrong, 1999; Colmer, 2003; Voesenek et al., 2006; Bailey-Serres and Voesenek, 2008; Licausi and Perata, 2009; Sauter, 2013; Voesenek and Bailey-Serres, 2015). To adapt to waterlogging in soil, rice (Oryza sativa) develops lysigenous aerenchyma in shoots (Matsukura et al., 2000; Colmer and Pedersen, 2008; Steffens et al., 2011) and roots (Jackson et al., 1985b; Justin and Armstrong, 1991; Kawai et al., 1998), which is formed by programmed cell death and subsequent lysis of some cortical cells (Jackson and Armstrong, 1999; Evans, 2004; Yamauchi et al., 2013). In rice roots, lysigenous aerenchyma is constitutively formed under aerobic conditions (Jackson et al., 1985b), and its formation is further induced under oxygen-deficient conditions (Colmer et al., 2006; Shiono et al., 2011). The former and latter are designated constitutive and inducible lysigenous aerenchyma formation, respectively (Colmer and Voesenek, 2009). The gaseous plant hormone ethylene regulates adaptive growth responses of plants to submergence (Voesenek and Blom, 1989; Voesenek et al., 1993; Visser et al., 1996a,b; Lorbiecke and Sauter, 1999; Hattori et al., 2009; Steffens and Sauter, 2009; van Veen et al., 2013). Ethylene also induces lysigenous aerenchyma formation in roots of some gramineous plants (Drew et al., 2000; Shiono et al., 2008). The treatment of roots with ethylene or its precursor (1-aminocyclopropane-1-carboxylic acid [ACC]) stimulates aerenchyma formation in rice (Justin and Armstrong, 1991; Colmer et al., 2006; Yukiyoshi and Karahara, 2014), maize (Zea mays; Drew et al., 1981; Jackson et al., 1985a; Takahashi et al., 2015), and wheat (Triticum aestivum; Yamauchi et al., 2014a,b). Moreover, treatment of roots with inhibitors of ethylene action or ethylene biosynthesis effectively blocks aerenchyma formation under hypoxic conditions in maize (Drew et al., 1981; Konings, 1982; Jackson et al., 1985a; Rajhi et al., 2011).Ethylene biosynthesis is accomplished by two main successive enzymatic reactions: conversion of S-adenosyl-Met to ACC by 1-aminocyclopropane-1-carboxylic acid synthase (ACS), and conversion of ACC to ethylene by 1-aminocyclopropane-1-carboxylic acid oxidase (ACO; Yang and Hoffman, 1984). The activities of both enzymes are enhanced during aerenchyma formation under hypoxic conditions in maize root (He et al., 1996). Since the ACC content in roots of maize is increased by oxygen deficiency and is strongly correlated with ethylene production (Atwell et al., 1988), ACC biosynthesis is essential for ethylene production during aerenchyma formation in roots. In fact, exogenously supplied ACC induced ethylene production in roots of maize (Drew et al., 1979; Konings, 1982; Atwell et al., 1988) and wheat (Yamauchi et al., 2014b), even under aerobic conditions. Ethylene production in plants is inversely related to oxygen concentration (Yang and Hoffman, 1984). Under anoxic conditions, the oxidation of ACC to ethylene by ACO, which requires oxygen, is almost completely repressed (Yip et al., 1988; Tonutti and Ramina, 1991). Indeed, anoxic conditions stimulate neither ethylene production nor aerenchyma formation in maize adventitious roots (Drew et al., 1979). Therefore, it is unlikely that the root tissues forming inducible aerenchyma are anoxic, and that the ACO-mediated step is repressed. Moreover, aerenchyma is constitutively formed in rice roots even under aerobic conditions (Jackson et al., 1985b), and thus, after the onset of waterlogging, oxygen can be immediately supplied to the apical regions of roots through the constitutively formed aerenchyma.Very-long-chain fatty acids (VLCFAs; ≥20 carbons) are major constituents of sphingolipids, cuticular waxes, and suberin in plants (Franke and Schreiber, 2007; Kunst and Samuels, 2009). In addition to their structural functions, VLCFAs directly or indirectly participate in several physiological processes (Zheng et al., 2005; Reina-Pinto et al., 2009; Roudier et al., 2010; Ito et al., 2011; Nobusawa et al., 2013; Tsuda et al., 2013), including the regulation of ethylene biosynthesis (Qin et al., 2007). During fiber cell elongation in cotton ovules, ethylene biosynthesis is enhanced by treatment with saturated VLCFAs, especially 24-carbon fatty acids, and is suppressed by an inhibitor of VLCFA biosynthesis (Qin et al., 2007). The first rate-limiting step in VLCFA biosynthesis is condensation of acyl-CoA with malonyl-CoA by β-ketoacyl-CoA synthase (KCS; Joubès et al., 2008). KCS enzymes are thought to determine the substrate and tissue specificities of fatty acid elongation (Joubès et al., 2008). The Arabidopsis (Arabidopsis thaliana) genome has 21 KCS genes (Joubès et al., 2008). In the Arabidopsis cut1 mutant, which has a defect in the gene encoding CUT1 that is required for cuticular wax production (i.e. one of the KCS genes), the expression of AtACO genes and growth of root cells were reduced when compared with the wild type (Qin et al., 2007). Furthermore, expression of the AtACO genes was rescued by exogenously supplied saturated VLCFAs (Qin et al., 2007). These observations imply that VLCFAs or their derivatives work as regulatory factors for gene expression during some physiological processes in plants.reduced culm number1 (rcn1) was first identified as a rice mutant with a low tillering rate in a paddy field (Takamure and Kinoshita, 1985; Yasuno et al., 2007). The rcn1 (rcn1-2) mutant has a single nucleotide substitution in the gene encoding a member of the ATP-binding cassette (ABC) transporter subfamily G, RCN1/OsABCG5, causing an Ala-684Pro substitution (Yasuno et al., 2009). The mutation results in several mutant phenotypes, although the substrates of RCN1/OsABCG5 have not been determined (Ureshi et al., 2012; Funabiki et al., 2013; Matsuda et al., 2014). We previously found that the rcn1 mutant has abnormal root morphology, such as shorter root length and brownish appearance of roots, under stagnant (deoxygenated) conditions (which mimics oxygen-deficient conditions in waterlogged soils). We also found that the rcn1 mutant accumulates less of the major suberin monomers originating from VLCFAs in the outer part of adventitious roots, and this results in a reduction of a functional apoplastic barrier in the root hypodermis (Shiono et al., 2014a).The objective of this study was to elucidate the molecular basis of inducible aerenchyma formation. To this end, we examined lysigenous aerenchyma formation and ACC, ethylene, and VLCFA accumulation and their biosyntheses in rcn1 roots. Based on the results of these studies, we propose that VLCFAs are involved in inducible aerenchyma formation through the enhancement of ethylene biosynthesis in rice roots.     

Sensitive UHPLC-MS/MS quantification method for 4β- and 4α-hydroxycholesterol in plasma for accurate CYP3A phenotyping

4β-Hydroxycholesterol (4β-OHC) is formed by Cytochrome P450 (CYP)3A and has drawn attention as an endogenous phenotyping probe for CYP3A activity. However, 4β-OHC is also increased by cholesterol autooxidation occurring in vitro due to dysregulated storage and in vivo by oxidative stress or inflammation, independent of CYP3A activity. 4α-hydroxycholesterol (4α-OHC), a stereoisomer of 4β-OHC, is also formed via autooxidation of cholesterol, not by CYP3A, and thus may have clinical potential in reflecting the state of cholesterol autooxidation. In this study, we establish a sensitive method for simultaneous quantification of 4β-OHC and 4α-OHC in human plasma using ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Plasma samples were prepared by saponification, two-step liquid-liquid extraction, and derivatization using picolinic acid. Intense [M+H]+ signals for 4β-OHC and 4α-OHC di-picolinyl esters were monitored using electrospray ionization. The assay fulfilled the requirements of the US Food and Drug Administration guidance for bioanalytical method validation, with a lower limit of quantification of 0.5 ng/ml for both 4β-OHC and 4α-OHC. Apparent recovery rates from human plasma ranged from 88.2% to 101.5% for 4β-OHC, and 91.8% to 114.9% for 4α-OHC. Additionally, matrix effects varied between 86.2% and 117.6% for 4β-OHC and between 89.5% and 116.9% for 4α-OHC. Plasma 4β-OHC and 4α-OHC concentrations in healthy volunteers, stage 3–5 chronic kidney disease (CKD) patients, and stage 5D CKD patients as measured by the validated assay were within the calibration ranges in all samples. We propose this novel quantification method may contribute to accurate evaluation of in vivo CYP3A activity.Supplementary key words: cholesterol, cytochrome P450, kidney, kinetics, pharmacokinetics, 4β-hydroxycholesterol, 4α-hydroxycholesterol, cytochrome P450 3A, mass spectrometry, plasma

Pharmacokinetics of drugs show large interindividual variability, and some drug-metabolizing enzymes and transporters are involved in the variability. Cytochrome P450 (CYP)3A is a major subfamily of metabolic enzymes involved in the metabolism of some drugs in the liver and small intestine (1). The main isoenzymes of this subfamily are CYP3A4 and CYP3A5. There is a large interindividual variability in CYP3A activity among patients, and the variability was reported to affect the clinical efficacy and the adverse reaction of CYP3A substrate drugs (2, 3). Thus, phenotyping of CYP3A activity is clinically important for more effective and safer treatment by CYP3A substrate drugs.Midazolam has been reported to be useful and considered a standard probe for CYP3A phenotyping (4, 5). Although midazolam is commonly used in drug-drug interaction studies (6, 7, 8, 9), this drug has some limitations in clinical application. For example, multiple blood samplings are needed to calculate the clearance for phenotyping, which limits its use in infants and elderly people. Midazolam shows high protein binding especially to albumin (approximately 96%) (10), and the free fraction may increase in patients with lower albumin levels, resulting in apparently increased hepatic clearance. Thus, phenotyping using midazolam may not be suitable in some patients with liver disease such as cirrhosis or kidney failure.To overcome these problems, 4β-hydroxycholesterol (4β-OHC) has drawn attention as an endogenous phenotyping probe for CYP3A activity. 4β-OHC is formed by CYP3A4 and CYP3A5 (11, 12) and has a long plasma half-life (approximately 17 days) (13). Since there is no circadian change in plasma 4β-OHC concentrations, one-point blood sampling is sufficient for CYP3A phenotyping. 4β-OHC is slowly metabolized by CYP7A1 (14), and CYP7A1 activity is not affected by kidney failure (15). Therefore, plasma 4β-OHC concentration is a suitable probe for CYP3A phenotyping in infants, elderly people, and patients with kidney failure or liver diseases including cirrhosis (16, 17, 18, 19, 20, 21).Several quantification methods have been reported for the measurement of plasma 4β-OHC concentrations using gas chromatography coupled to mass spectrometry (11) and high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) (22, 23, 24, 25, 26). Recently, Hautajärvi et al. (27) reported an ultra-high performance liquid chromatography coupled to high resolution mass spectrometry method for quantification of plasma 4β-OHC and 4α-hydroxycholesterol (4α-OHC) concentrations. 4α-OHC, a stereoisomer of 4β-OHC, is formed via autooxidation of cholesterol, and not by CYP3A. Therefore, plasma 4α-OHC concentration reflects plasma sample stability, because plasma 4α-OHC concentration increases in uncontrolled storage condition (28). Furthermore, oxysterols including 4β-OHC and 4α-OHC have been reported to be elevated by cholesterol autoxidation due to oxidative stress or inflammation in the liver, regardless of CYP3A activity (29). Thus, simultaneous quantification of 4β-OHC and 4α-OHC is preferred for phenotyping of CYP3A activity using clinical plasma samples.In this study, we established a sensitive method for simultaneous quantification of 4β-OHC and 4α-OHC in human plasma using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The method was applied to measure plasma 4β-OHC and 4α-OHC concentrations in healthy volunteers and patients with chronic kidney disease (CKD).     

Sterol-Dependent Induction of Plant Defense Responses by a Microbe-Associated Molecular Pattern from Trichoderma viride

Plant-microbe interactions involve numerous regulatory systems essential for plant defense against pathogens. An ethylene-inducing xylanase (Eix) of Trichoderma viride is a potent elicitor of plant defense responses in specific cultivars of tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum). We demonstrate that tomato cyclopropyl isomerase (SlCPI), an enzyme involved in sterol biosynthesis, interacts with the LeEix2 receptor. Moreover, we examined the role of SlCPI in signaling during the LeEix/Eix defense response. We found that SlCPI is an important factor in the regulation of the induction of defense responses such as the hypersensitive response, ethylene biosynthesis, and the induction of pathogenesis-related protein expression in the case of LeEix/Eix. Our results also suggest that changes in the sterol composition reduce LeEix internalization, thereby attenuating the induction of plant defense responses.Plant innate immunity is activated upon the recognition of pathogen- and microbe-associated molecular patterns by surface-localized immune receptors or the stimulation of cytoplasmic immune receptors by pathogen effector proteins (Jones and Dangl, 2006; Thomma et al., 2011). Leucine-rich repeat (LRR) receptor kinases and leucine-rich repeat receptor proteins (LRR-RLPs) respond to conserved microbe-associated molecular patterns by producing a defense response upon detection (Altenbach and Robatzek, 2007; Bittel and Robatzek, 2007; Robatzek et al., 2007; Geldner and Robatzek, 2008). One such LRR-RLP is the ethylene-inducing xylanase (Eix) receptor LeEix2. The fungal protein Eix (Dean et al., 1989) is a well-known protein elicitor of defense response reactions in tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum; Bailey et al., 1990; Avni et al., 1994). Eix induces ethylene biosynthesis, extensive electrolyte leakage, pathogenesis-related (PR) gene expression, reactive oxygen species (ROS), and the hypersensitive response (HR; Bailey et al., 1990; Ron et al., 2000). Eix was shown to specifically bind to the plasma membrane of responsive cultivars of both tomato and tobacco (Hanania and Avni, 1997). The response to Eix in tobacco and tomato cultivars is controlled by an LRR-RLP encoded by a single locus, termed LeEix (Ron and Avni, 2004). Previously, we showed that Eix triggers internalization of the LeEix2 receptor and its localization to endosomes (Bar and Avni, 2009).Endocytic processes and vesicular transport in general require the participation of membrane components that form transport vesicles with a capability to store and process a number of molecules known to participate in cell signaling (Anderson, 1993; Patel et al., 2008; Hansen and Nichols, 2010). Sterols are lipophilic membrane components that have many important functions in an array of eukaryotes. Changes in membrane-bound sterol levels and composition can have effects on the activity of membrane proteins and on signal transduction processes. The interaction between sterols and phospholipids forms microdomains termed lipid rafts (Simons and Ikonen, 1997). In response to cellular stimuli, lipid rafts can change the protein microenvironment, leading to the initiation of signaling cascades (Simons and Toomre, 2000; Mongrand et al., 2010; Simon-Plas et al., 2011). Sterols also provide precursors for the biosynthesis of steroid hormones such as mammalian estrogens and glucocorticoids and plant brassinosteroids (Bishop and Yokota, 2001; Benveniste, 2004; Suzuki and Muranaka, 2007). One of the enzymes involved in sterol biosynthesis is cyclopropyl isomerase (CPI; Lovato et al., 2000; Benveniste, 2004).A variety of endocytic pathways have been described in mammalian and fungal cells that differ mainly in the size, shape, and composition of endocytic vesicles and in the participation of different proteins (Conner and Schmid, 2003; Soldati and Schliwa, 2006). Cholesterol, the main mammalian sterol, has an important role in most internalization steps through both caveolae and clathrin-coated pits (Murata et al., 1995; Subtil et al., 1999). Cholesterol depletion alters endocytic structures and reduces the polar delivery of target proteins (Keller and Simons, 1998; Pichler and Riezman, 2004). Plant sterols are reported to be internalized into endosomes and distributed throughout the endocytic pathway in an actin-dependent manner (Grebe et al., 2003). The sterol endocytic pathway has been shown to interrupt the internalization, trafficking, and polar recycling of PIN2, an auxin efflux facilitator and polarity marker, in developing root epidermal cells of Arabidopsis (Arabidopsis thaliana; Grebe et al., 2003; Men et al., 2008). Sterols were shown to function in the trafficking of an ATP-binding cassette (ABCB19) from the trans-Golgi to the plasma membrane (Yang et al., 2013).Here, we report the isolation of SlCPI, which interacts with the LeEix2 receptor. Modulating the expression or function of SlCPI affects the induction of plant defense responses mediated by Eix.     

CELLULOSE SYNTHASE-LIKE A2, a Glucomannan Synthase,Is Involved in Maintaining Adherent Mucilage Structure in Arabidopsis Seed

Mannans are hemicellulosic polysaccharides that are considered to have both structural and storage functions in the plant cell wall. However, it is not yet known how mannans function in Arabidopsis (Arabidopsis thaliana) seed mucilage. In this study, CELLULOSE SYNTHASE-LIKE A2 (CSLA2; At5g22740) expression was observed in several seed tissues, including the epidermal cells of developing seed coats. Disruption of CSLA2 resulted in thinner adherent mucilage halos, although the total amount of the adherent mucilage did not change compared with the wild type. This suggested that the adherent mucilage in the mutant was more compact compared with that of the wild type. In accordance with the role of CSLA2 in glucomannan synthesis, csla2-1 mucilage contained 30% less mannosyl and glucosyl content than did the wild type. No appreciable changes in the composition, structure, or macromolecular properties were observed for nonmannan polysaccharides in mutant mucilage. Biochemical analysis revealed that cellulose crystallinity was substantially reduced in csla2-1 mucilage; this was supported by the removal of most mucilage cellulose through treatment of csla2-1 seeds with endo-β-glucanase. Mutation in CSLA2 also resulted in altered spatial distribution of cellulose and an absence of birefringent cellulose microfibrils within the adherent mucilage. As with the observed changes in crystalline cellulose, the spatial distribution of pectin was also modified in csla2-1 mucilage. Taken together, our results demonstrate that glucomannans synthesized by CSLA2 are involved in modulating the structure of adherent mucilage, potentially through altering cellulose organization and crystallization.Mannan polysaccharides are a complex set of hemicellulosic cell wall polymers that are considered to have both structural and storage functions. Based on the particular chemical composition of the backbone and the side chains, mannan polysaccharides are classified into four types: pure mannan, glucomannan, galactomannan, and galactoglucomannan (Moreira and Filho, 2008; Wang et al., 2012; Pauly et al., 2013). Each of these polysaccharides is composed of a β-1,4-linked backbone containing Man or a combination of Glc and Man residues. In addition, the mannan backbone can be substituted with side chains of α-1,6-linked Gal residues. Mannan polysaccharides have been proposed to cross link with cellulose and other hemicelluloses via hydrogen bonds (Fry, 1986; Iiyama et al., 1994; Obel et al., 2007; Scheller and Ulvskov, 2010). Furthermore, it has been reported that heteromannans with different levels of substitution can interact with cellulose in diverse ways (Whitney et al., 1998). Together, these observations indicate the complexity of mannan polysaccharides in the context of cell wall architecture.CELLULOSE SYNTHASE-LIKE A (CSLA) enzymes have been shown to have mannan synthase activity in vitro. These enzymes polymerize the β-1,4-linked backbone of mannans or glucomannans, depending on the substrates (GDP-Man and/or GDP-Glc) provided (Richmond and Somerville, 2000; Liepman et al., 2005, 2007; Pauly et al., 2013). In Arabidopsis (Arabidopsis thaliana), nine CSLA genes have been identified; different CSLAs are responsible for the synthesis of different mannan types (Liepman et al., 2005, 2007). CSLA7 has mannan synthase activity in vitro (Liepman et al., 2005) and has been shown to synthesize stem glucomannan in vivo (Goubet et al., 2009). Disrupting the CSLA7 gene results in defective pollen growth and embryo lethality phenotypes in Arabidopsis, indicating structural or signaling functions of mannan polysaccharides during plant embryo development (Goubet et al., 2003). A mutation in CSLA9 results in the inhibition of Agrobacterium tumefaciens-mediated root transformation in the rat4 mutant (Zhu et al., 2003). CSLA2, CSLA3, and CSLA9 are proposed to play nonredundant roles in the biosynthesis of stem glucomannans, although mutations in CSLA2, CSLA3, or CSLA9 have no effect on stem development or strength (Goubet et al., 2009). All of the Arabidopsis CSLA proteins have been shown to be involved in the biosynthesis of mannan polysaccharides in the plant cell wall (Liepman et al., 2005, 2007), although the precise physiological functions of only CSLA7 and CSLA9 have been conclusively demonstrated.In Arabidopsis, when mature dry seeds are hydrated, gel-like mucilage is extruded to envelop the entire seed. Ruthenium red staining of Arabidopsis seeds reveals two different mucilage layers, termed the nonadherent and the adherent mucilage layers (Western et al., 2000; Macquet et al., 2007a). The outer, nonadherent mucilage is loosely attached and can be easily extracted by shaking seeds in water. Compositional and linkage analyses suggest that this layer is almost exclusively composed of unbranched rhamnogalacturonan I (RG-I) (>80% to 90%), with small amounts of branched RG-I, arabinoxylan, and high methylesterified homogalacturonan (HG). By contrast, the inner, adherent mucilage layer is tightly attached to the seed and can only be removed by strong acid or base treatment, or by enzymatic digestion (Macquet et al., 2007a; Huang et al., 2011; Walker et al., 2011). As with the nonadherent layer, adherent mucilage is also mainly composed of unbranched RG-I, but with small numbers of arabinan and galactan ramifications (Penfield et al., 2001; Willats et al., 2001; Dean et al., 2007; Macquet et al., 2007a, 2007b; Arsovski et al., 2009; Haughn and Western, 2012). There are also minor amounts of pectic HG in the adherent mucilage, with high methylesterified HG in the external domain compared with the internal domain of the adherent layer (Willats et al., 2001; Macquet et al., 2007a; Rautengarten et al., 2008; Sullivan et al., 2011; Saez-Aguayo et al., 2013). In addition, the adherent mucilage contains cellulose (Blake et al., 2006; Macquet et al., 2007a), which is entangled with RG-I and is thought to anchor the pectin-rich mucilage onto seeds (Macquet et al., 2007a; Harpaz-Saad et al., 2011, 2012; Mendu et al., 2011; Sullivan et al., 2011). As such, Arabidopsis seed mucilage is considered to be a useful model for investigating the biosynthesis of cell wall polysaccharides and how this process is regulated in vivo (Haughn and Western, 2012).Screening for altered seed coat mucilage has led to the identification of several genes encoding enzymes that are involved in the biosynthesis or modification of mucilage components. RHAMNOSE SYNTHASE2/MUCILAGE-MODIFIED4 (MUM4) is responsible for the synthesis of UDP-l-Rha (Usadel et al., 2004; Western et al., 2004; Oka et al., 2007). The putative GALACTURONSYLTRANSFERASE11 can potentially synthesize mucilage RG-I or HG pectin from UDP-d-GalUA (Caffall et al., 2009). GALACTURONSYLTRANSFERASE-LIKE5 appears to function in the regulation of the final size of the mucilage RG-I (Kong et al., 2011, 2013). Mutant seeds defective in these genes display reduced thickness of the extruded mucilage layer compared with wild-type Arabidopsis seeds.RG-I deposited in the apoplast of seed coat epidermal cells appears to be synthesized in a branched form that is subsequently modified by enzymes in the apoplast. MUM2 encodes a β-galactosidase that removes Gal residues from RG-I side chains (Dean et al., 2007; Macquet et al., 2007b). β-XYLOSIDASE1 encodes an α-l-arabinfuranosidase that removes Ara residues from RG-I side chains (Arsovski et al., 2009). Disruptions of these genes lead to defective hydration properties and affect the extrusion of mucilage. Furthermore, correct methylesterification of mucilage HG is also required for mucilage extrusion. HG is secreted into the wall in a high methylesterified form that can then be enzymatically demethylesterified by pectin methylesterases (PMEs; Bosch and Hepler, 2005). PECTIN METHYLESTERASE INHIBITOR6 (PMEI6) inhibits PME activities (Saez-Aguayo et al., 2013). The subtilisin-like Ser protease (SBT1.7) can activate other PME inhibitors, but not PMEI6 (Rautengarten et al., 2008; Saez-Aguayo et al., 2013). Disruption of either PMEI6 or SBT1.7 results in the delay of mucilage release.Although cellulose is present at low levels in adherent mucilage, it plays an important adhesive role for the attachment of mucilage pectin to the seed coat epidermal cells. The orientation and amount of pectin associated with the cellulose network is largely determined by cellulose conformation properties (Macquet et al., 2007a; Haughn and Western, 2012). Previous studies have demonstrated that CELLULOSE SYNTHASE A5 (CESA5) is required for the production of seed mucilage cellulose and the adherent mucilage in the cesa5 mutant can be easily extracted with water (Harpaz-Saad et al., 2011, 2012; Mendu et al., 2011; Sullivan et al., 2011).Despite all of these discoveries, large gaps remain in the current knowledge of the biosynthesis and functions of mucilage polysaccharides in seed coats. In this study, we show that CSLA2 is involved in the biosynthesis of mucilage glucomannan. Furthermore, we show that CSLA2 functions in the maintenance of the normal structure of the adherent mucilage layer through modifying the mucilage cellulose ultrastructure.