Focus on Metabolism: Glutathionylation and Reduction of Methacrolein in Tomato Plants Account for Its Absorption from the Vapor Phase

A large portion of the volatile organic compounds emitted by plants are oxygenated to yield reactive carbonyl species, which have a big impact on atmospheric chemistry. Deposition to vegetation driven by the absorption of reactive carbonyl species into plants plays a major role in cleansing the atmosphere, but the mechanisms supporting this absorption have been little examined. Here, we performed model experiments using methacrolein (MACR), one of the major reactive carbonyl species formed from isoprene, and tomato (Solanum lycopersicum) plants. Tomato shoots enclosed in a jar with MACR vapor efficiently absorbed MACR. The absorption efficiency was much higher than expected from the gas/liquid partition coefficient of MACR, indicating that MACR was likely metabolized in leaf tissues. Isobutyraldehyde, isobutyl alcohol, and methallyl alcohol (MAA) were detected in the headspace and inside tomato tissues treated with MACR vapor, suggesting that MACR was enzymatically reduced. Glutathione (GSH) conjugates of MACR (MACR-GSH) and MAA (MAA-GSH) were also detected. MACR-GSH was essentially formed through spontaneous conjugation between endogenous GSH and exogenous MACR, and reduction of MACR-GSH to MAA-GSH was likely catalyzed by an NADPH-dependent enzyme in tomato leaves. Glutathionylation was the metabolic pathway most responsible for the absorption of MACR, but when the amount of MACR exceeded the available GSH, MACR that accumulated reduced photosynthetic capacity. In an experiment simulating the natural environment using gas flow, MACR-GSH and MAA-GSH accumulation accounted for 30% to 40% of the MACR supplied. These results suggest that MACR metabolism, especially spontaneous glutathionylation, is an essential factor supporting MACR absorption from the atmosphere by tomato plants.Plants emit vast amounts of volatile organic chemicals (VOCs) into the atmosphere. The annual emission of VOCs other than methane is estimated to be approximately 1,300 Tg of carbon (Goldstein and Galbally, 2007), with approximately 90% originating from biogenic sources, of which one-third (approximately 500 Tg of carbon/year) is isoprene (Guenther et al., 1995). In the atmosphere, VOCs undergo the chemical processes of photolysis and reaction with hydroxyl and nitrate radicals (Atkinson and Arey, 2003). Isoprene, for example, is converted into a series of isomeric hydroxyl-substituted alkyl peroxyl radicals, which are further converted into methyl vinyl ketone (MVK; but-3-en-2-one) and methacrolein (MACR; 2-methylprop-2-enal; Liu et al., 2013). These VOCs and their oxygenated products (oVOCs) are important components for the production of ozone and aerosols, and thus have a big impact on atmospheric chemistry and even on the climate system (Goldstein and Galbally, 2007). VOCs and oVOCs are removed from the atmosphere through oxidation to carbon monoxide or dioxide, dry or wet deposition, or secondary aerosol formation (Goldstein and Galbally, 2007). Among these, deposition to vegetation plays a major role in the removal of VOCs and oVOCs from the atmosphere (Karl et al., 2010).A significant portion of the deposition to vegetation is attributable to the uptake of VOCs and oVOCs by plants, and a field study showed that MVK and MACR were immediately lost once they entered a leaf through stomata (Karl et al., 2010). Under growth conditions where stomatal conductance is high enough, the partitioning of VOCs between air and leaf water phases in equilibrium and the capacity of the plant to metabolize, translocate, and store VOCs determine their uptake rate (Tani et al., 2013). The immediate loss in leaves observed with MVK and MACR is indicative of efficient enzymatic reactions metabolizing them; however, the details of the metabolism of these oVOCs have been little investigated so far.The absorption and metabolism of several VOCs by plants have been reported. Airborne ent-kaurene was absorbed by Arabidopsis (Arabidopsis thaliana), Japanese cypress (Chamaecyparis obtusa), and Japanese cedar (Cryptomeria japonica) plants and converted into GAs (Otsuka et al., 2004). Arabidopsis absorbed (Z)-3-hexenal and converted it into (Z)-3-hexen-1-ol or further into (Z)-3-hexen-1-yl acetate using NADPH and acetyl-CoA, probably inside the plant tissues (Matsui et al., 2012). Nicotiana attenuata plants absorbed dimethyl disulfide formed by rhizobacteria (Meldau et al., 2013). The sulfur atom derived from volatile dimethyl disulfide was assimilated into plant proteins. Karl et al. (2010) assumed that aldehyde dehydrogenase, which is involved in detoxification that limits aldehyde accumulation and oxidative stress (Kirch et al., 2004), is involved in the uptake of oVOCs containing an aldehyde moiety; however, they did not provide direct evidence supporting their assumption.Conjugation of VOCs and oVOCs with sugar or glutathione (GSH) is another way to metabolize them. (Z)-3-Hexen-1-ol in the vapor phase was taken up by tomato (Solanum lycopersicum) plants and converted into its glycoside (Sugimoto et al., 2014). (E)-2-Hexenal reacts with GSH spontaneously and/or via glutathione S-transferase (GST) to form hexanal-GSH, which is subsequently reduced to hexanol-GSH (Davoine et al., 2006), although it is uncertain whether airborne (E)-2-hexenal is converted into its corresponding GSH adduct. Glutathionylation of (E)-2-hexenal is common and has been confirmed in grapevine (Vitis vinifera) and passion fruit (Passiflora edulis; Kobayashi et al., 2011; Fedrizzi et al., 2012). The catabolites formed from the GSH adduct in these crops are precursors for important flavor components.Although it is clear that oVOCs are absorbed by vegetation and that their efficient uptake is probably supported by metabolism in plant tissues, the metabolic fates of oVOCs taken up from the vapor phase into plants have been little studied. Here, we performed a series of model experiments using tomato seedlings and MACR to dissect the fates of oVOCs once they entered into plant tissues. To clearly see absorption of MACR and its fates in plant tissues, a model experiment under enclosed conditions with a high concentration of MACR was first carried out. Subsequently, an airflow system with a realistically low concentration of MACR was used. Tomato plants efficiently absorbed MACR. Reduction of the carbonyl moiety and the double bond conjugated to the carbonyl and conjugation with GSH were the major methods of metabolism of exogenous MACR. The metabolism seemed to be involved in the detoxification of reactive carbonyl species, which, in turn, accounted for the oVOC deposition to vegetation.     

中国西北地区中国林蛙各居群的分类学研究(两栖纲:蛙科)

采用形态学比较和数值分类相结合,对中国西北部分布地中国林蛙Ｒａｎａ ｃｈｅｎｓｉｎｅｎｓｉｓ各居群间（包括分布于青藏庙的地区的原定种或亚种）的差差水平进行了探讨。结果认为：分布于中国西北部高原地区的各居群其形态特征和数值分析这〖其中包括原定名为Ｒａｎａ ａｍｕｒｅｎｓｉｓ ｋｕｋｕｎｏｒｉｓ（Ｎｉｋｏｌ ｓｋｉｉ,１９１８,青海湖）,Ｒ．ｗｅｉｇｏｌｉｄ（Ｖｏｇｔ,１９２４,四川甘孜和德格）,Ｒ．     

Rum1, an inhibitor of cyclin-dependent kinase in fission yeast, is negatively regulated by mitogen-activated protein kinase-mediated phosphorylation at Ser and Thr residues.

The p25(rum1) is an inhibitor of Cdc2 kinase expressed in fission yeast and plays an important role in cell-cycle control. As its amino-acid sequence suggests that p25(rum1) has putative phosphorylation sites for mitogen-activated protein kinase (MAPK), we investigated the ability of MAPK to phosphorylate p25(rum1). Direct in vitro kinase assay using GST-fusion proteins of wild-type as well as various mutants of p25(rum1) demonstrated that MAPK phosphorylates the N-terminal portion of p25(rum1) and residues Thr13 and Ser19 are major phosphorylation sites for MAPK. In addition, phosphorylation of p25(rum1) by MAPK revealed markedly reduced Cdc2 kinase inhibitor ability of the protein. Together with the fact that replacement of both Thr13 and Ser19 with Glu, which mimics the phosphorylated state of these residues, also significantly reduces the activity of p25(rum1) as a Cdc2 inhibitor, it was suggested that the phosphorylation of Thr13 and Ser19 negatively regulates the function of p25(rum1). Further evidence indicates that phosphorylation of Thr13 and Ser19 may retain a negative effect on the function of p25(rum1) even in vivo. Therefore, MAPK may regulate the function of p25(rum1) via phosphorylation of its Thr and Ser residues and thus participate in cell cycle control in fission yeast.     

Regulation of N-methyl-D-aspartate receptors by calpain in cortical neurons

The N-methyl-D-aspartate (NMDA) receptor is a cation channel highly permeable to calcium and plays critical roles in governing normal and pathologic functions in neurons. Calcium entry through NMDA receptors (NMDARs) can lead to the activation of the Ca2+-dependent protease, calpain. Here we investigated the involvement of calpain in regulation of NMDAR channel function. After prolonged (5-min) treatment with NMDA or glutamate, the whole-cell NMDAR-mediated current was significantly reduced in both acutely dissociated and cultured cortical pyramidal neurons. The down-regulation of NMDAR current was blocked by bath application of selective calpain inhibitors. Intracellular injection of a specific calpain inhibitory peptide also eliminated the down-regulation of NMDAR current induced by prolonged NMDA treatment. In contrast, dynamin inhibitory peptide had no effect on the depression of NMDAR current, suggesting the lack of involvement of dynamin/clathrin-mediated NMDAR internalization in this process. Immunoblotting analysis showed that the NR2A and NR2B subunits of NMDARs were markedly degraded in cultured cortical neurons treated with glutamate, and the degradation of NR2 subunits was prevented by calpain inhibitors. Taken together, our results suggest that prolonged activation of NMDARs in neurons activates calpain, and activated calpain in turn down-regulates the function of NMDARs, which provides a neuroprotective mechanism against NMDAR overstimulation accompanying ischemia and stroke.     

Lysophosphatidylcholine enhances glucose-dependent insulin secretion via an orphan G-protein-coupled receptor

A lysophospholipid series, such as lysophosphatidic acid, lysophosphatidylserine, and lysophosphatidylcholine (LPC), is a bioactive lipid mediator with diverse physiological and pathological functions. LPC has been reported to induce insulin secretion from pancreatic beta-cells, however, the precise mechanism has remained elusive to date. Here we show that an orphan G-protein-coupled receptor GPR119 plays a pivotal role in this event. LPC potently enhances insulin secretion in response to high concentrations of glucose in the perfused rat pancreas via stimulation of adenylate cyclase, and dose-dependently induces intracellular cAMP accumulation and insulin secretion in a mouse pancreatic beta-cell line, NIT-1 cells. The Gs-protein-coupled receptor for LPC was identified as GPR119, which is predominantly expressed in the pancreas. GPR119-specific siRNA significantly blocked LPC-induced insulin secretion from NIT-1 cells. Our findings suggest that GPR119, which is a novel endogenous receptor for LPC, is involved in insulin secretion from beta-cells, and is a potential target for anti-diabetic drug development.     

CLASP1 and CLASP2 bind to EB1 and regulate microtubule plus-end dynamics at the cell cortex

CLIP-associating protein (CLASP) 1 and CLASP2 are mammalian microtubule (MT) plus-end binding proteins, which associate with CLIP-170 and CLIP-115. Using RNA interference in HeLa cells, we show that the two CLASPs play redundant roles in regulating the density, length distribution and stability of interphase MTs. In HeLa cells, both CLASPs concentrate on the distal MT ends in a narrow region at the cell margin. CLASPs stabilize MTs by promoting pauses and restricting MT growth and shortening episodes to this peripheral cell region. We demonstrate that the middle part of CLASPs binds directly to EB1 and to MTs. Furthermore, we show that the association of CLASP2 with the cell cortex is MT independent and relies on its COOH-terminal domain. Both EB1- and cortex-binding domains of CLASP are required to promote MT stability. We propose that CLASPs can mediate interactions between MT plus ends and the cell cortex and act as local rescue factors, possibly through forming a complex with EB1 at MT tips.     

Autoantibodies to T cell costimulatory molecules in systemic autoimmune diseases

To determine whether antilymphocyte Abs to T cell costimulatory molecules are generated in patients with autoimmune diseases and, if they exist, to clarify the mechanism of their production and pathological roles, we investigated the presence of autoantibodies to CTLA-4 (CD152), CD28, B7-1 (CD80), and B7-2 (CD86) in serum samples obtained from patients with various autoimmune diseases and from normal subjects using recombinant fusion proteins. In ELISAs, anti-CD28, anti-B7-1, and anti-B7-2 Abs were rarely seen, whereas anti-CTLA-4 Abs were detected in 8.2% of the patients with systemic lupus erythematosus, 18.8% of those with rheumatoid arthritis, 3.1% of those with systemic sclerosis, 31.8% of those with Beh?et's disease, 13.3% of those with Sj?gren's syndrome, and 0% of healthy donors. This reactivity was confirmed by immunoblotting. More importantly, the purified anti-CTLA-4 Abs reacted with CTLA-4 expressed on P815 cells by flow cytometry. In addition, we found at least three epitopes on the CTLA-4 molecule. Furthermore, among the patients with Beh?et's disease, uveitis was seen significantly less frequently in the anti-CTLA-4 Ab-positive patients. Taken collectively, these data indicate that anti-CTLA-4 autoantibodies are generated in systemic autoimmune diseases by an Ag-driven mechanism and may modulate the immune response in vivo by binding to CTLA-4 on T cells.     

Sequence and expression of the mRNA encoding the chymotrypsin inhibitor in winged bean (Psophocarpus tetragonolobus (L.) DC.)

The accumulation of the Kunitz-type chymotrypsin inhibitor WCI-3 in winged bean seeds is controlled developmentally. In vitro translation experiments showed that the WCI-3 mRNA was present in 35- and 40-day-old immature seeds after flowering. The size of the in vitro translation product is about 2 000 Da larger than that of the mature WCI-3 protein. The WCI-3 cDNA clones were isolated from a gtll cDNA library of 35-day-old immature seeds by immunoscreening. A nearly full-length cDNA clone was obtained containing an open reading frame of 207 amino acid residues. The deduced sequence of the 183 carboxy terminal amino acids coincides precisely with the amino acid sequence determined for purified WCI-3. The amino terminal extension of 24 residues has the characteristics of a signal peptide. Northern hybridization analysis of total poly(A)⁺ RNA showed that the WCI-3 mRNA is approximately 900 nucleotides long and accumulates in 35- and 40-day-old but not in 30-day-old immature seeds.     

Canonical Wnt signaling and its antagonist regulate anterior-posterior axis polarization by guiding cell migration in mouse visceral endoderm

The mouse embryonic axis is initially formed with a proximal-distal orientation followed by subsequent conversion to a prospective anterior-posterior (A-P) polarity with directional migration of visceral endoderm cells. Importantly, Otx2, a homeobox gene, is essential to this developmental process. However, the genetic regulatory mechanism governing axis conversion is poorly understood. Here, defective axis conversion due to Otx2 deficiency can be rescued by expression of Dkk1, a Wnt antagonist, or following removal of one copy of the beta-catenin gene. Misexpression of a canonical Wnt ligand can also inhibit correct A-P axis rotation. Moreover, asymmetrical distribution of beta-catenin localization is impaired in the Otx2-deficient and Wnt-misexpressing visceral endoderm. Concurrently, canonical Wnt and Dkk1 function as repulsive and attractive guidance cues, respectively, in the migration of visceral endoderm cells. We propose that Wnt/beta-catenin signaling mediates A-P axis polarization by guiding cell migration toward the prospective anterior in the pregastrula mouse embryo.