Regulation of epinasty induced by 2,4‐dichlorophenoxyacetic acid in pea and Arabidopsis plants

The herbicide 2,4‐dichlorophenoxyacetic acid (2,4‐D) causes uncontrolled cell division and malformed growth in plants, giving rise to leaf epinasty and stem curvature. In this study, mechanisms involved in the regulation of leaf epinasty induced by 2,4‐D were studied using different chemicals involved in reactive oxygen species (ROS) accumulation (diphenyleniodonium, butylated hydroxyanisole, EDTA, allopurinol), calcium channels (LaCl₃), protein phosphorylation (cantharidin, wortmannin) and ethylene emission/perception (aminoethoxyvinyl glycine, AgNO₃). The effect of these compounds on the epinasty induced by 2,4‐D was analysed in shoots and leaf strips from pea plants. For further insight into the effect of 2,4‐D, studies were also made in Arabidopsis mutants deficient in ROS production (rbohD, rbohF, xdh), ethylene (ein 3‐1, ctr 1‐1, etr 1‐1), abscisic acid (aba 3.1), and jasmonic acid (coi 1.1, jar 1.1, opr 3) pathways. The results suggest that ROS production, mainly ·OH, is essential in the development of epinasty triggered by 2,4‐D. Epinasty was also found to be regulated by Ca²⁺, protein phosphorylation and ethylene, although all these factors act downstream of ROS production. The use of Arabidopsis mutants appears to indicate that abscisic and jasmonic acid are not involved in regulating epinasty, although they could be involved in other symptoms induced by 2,4‐D.     

Large‐scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis

Protein phosphorylation regulates a wide range of cellular processes. Here, we report the proteome‐wide mapping of in vivo phosphorylation sites in Arabidopsis by using complementary phosphopeptide enrichment techniques coupled with high‐accuracy mass spectrometry. Using unfractionated whole cell lysates of Arabidopsis, we identified 2597 phosphopeptides with 2172 high‐confidence, unique phosphorylation sites from 1346 proteins. The distribution of phosphoserine, phosphothreonine, and phosphotyrosine sites was 85.0, 10.7, and 4.3%. Although typical tyrosine‐specific protein kinases are absent in Arabidopsis, the proportion of phosphotyrosines among the phospho‐residues in Arabidopsis is similar to that in humans, where over 90 tyrosine‐specific protein kinases have been identified. In addition, the tyrosine phosphoproteome shows features distinct from those of the serine and threonine phosphoproteomes. Taken together, we highlight the extent and contribution of tyrosine phosphorylation in plants.     

The grapevine (Vitis vinifera) LysM receptor kinases VvLYK1‐1 and VvLYK1‐2 mediate chitooligosaccharide‐triggered immunity

Chitin, a major component of fungal cell walls, is a well‐known pathogen‐associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM‐RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM‐RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin‐induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1‐1, ‐2, or ‐3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1‐1 and VvLYK1‐2, but not VvLYK1‐3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide‐induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1‐1 in Atcerk1 restored penetration resistance to the non‐adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1‐1 and VvLYK1‐2 participate in chitin‐ and chitosan‐triggered immunity and that VvLYK1‐1 plays an important role in basal resistance against E. necator.     

Ethylene and salicylic acid control glutathione biosynthesis in ozone‐exposed Arabidopsis thaliana

Ozone produces reactive oxygen species and induces the synthesis of phytohormones, including ethylene and salicylic acid. These phytohormones act as signal molecules that enhance cell death in response to ozone exposure. However, some studies have shown that ethylene and salicylic acid can instead decrease the magnitude of ozone‐induced cell death. Therefore, we studied the defensive roles of ethylene and salicylic acid against ozone. Unlike the wild‐type, Col‐0, Arabidopsis mutants deficient in ethylene signaling (ein2) or salicylic acid biosynthesis (sid2) generated high levels of superoxide and exhibited visible leaf injury, indicating that ethylene and salicylic acid can reduce ozone damage. Macroarray analysis suggested that the ethylene and salicylic acid defects influenced glutathione (GSH) metabolism. Increases in the reduced form of GSH occurred in Col‐0 6 h after ozone exposure, but little GSH was detected in ein2 and sid2 mutants, suggesting that GSH levels were affected by ethylene or salicylic acid signaling. We performed gene expression analysis by real‐time polymerase chain reaction using genes involved in GSH metabolism. Induction of γ‐glutamylcysteine synthetase (GSH1), glutathione synthetase (GSH2), and glutathione reductase 1 (GR1) expression occurred normally in Col‐0, but at much lower levels in ein2 and sid2. Enzymatic activities of GSH1 and GSH2 in ein2 and sid2 were significantly lower than in Col‐0. Moreover, ozone‐induced leaf damage observed in ein2 and sid2 was mitigated by artificial elevation of GSH content. Our results suggest that ethylene and salicylic acid protect against ozone‐induced leaf injury by increasing de novo biosynthesis of GSH.     

The Myo‐inositol pathway does not contribute to ascorbic acid synthesis

• Ascorbic acid (AsA) biosynthesis in plants predominantly occurs via a pathway with d ‐mannose and l ‐galactose as intermediates. One alternative pathway for AsA synthesis, which is similar to the biosynthesis route in mammals, is controversially discussed for plants. Here, myo‐inositol is cleaved to glucuronic acid and then converted via l ‐gulonate to AsA. In contrast to animals, plants have an effective recycling pathway for glucuronic acid, being a competitor for the metabolic rate. Recycling involves a phosphorylation at C1 by the enzyme glucuronokinase.
• Two previously described T‐DNA insertion lines in the gene coding for glucuronokinase1 show wild type‐like expression levels of the mRNA in our experiments and do not accumulate glucuronic acid in labelling experiments disproving that these lines are true knockouts. As suitable T‐DNA insertion lines were not available, we generated frameshift mutations in the major expressed isoform glucuronokinase1 (At3g01640) to potentially redirect metabolites to AsA.
• However, radiotracer experiments with ³H‐myo‐inositol revealed that the mutants in glucuronokinase1 accumulate only glucuronic acid and incorporate less metabolite into cell wall polymers. AsA was not labelled, suggesting that Arabidopsis cannot efficiently use glucuronic acid for AsA biosynthesis.
• All four mutants in glucuronokinase as well as the wild type have the same level of AsA in leaves.

     

Association of cytochrome b5 with ETR1 ethylene receptor signaling through RTE1 in Arabidopsis

Ethylene plays important roles in plant growth, development and stress responses, and is perceived by a family of receptors that repress ethylene responses when ethylene is absent. Repression by the ethylene receptor ETR1 depends on an integral membrane protein, REVERSION TO ETHYLENE SENSITIVITY1 (RTE1), which acts upstream of ETR1 in the endoplasmic reticulum (ER) membrane and Golgi apparatus. To investigate RTE1 function, we screened for RTE1‐interacting proteins using the yeast split‐ubiquitin assay, which yielded the ER‐localized cytochrome b₅ (Cb5) isoform D. Cb5s are small hemoproteins that perform electron transfer reactions in all eukaryotes, but their roles in plants are relatively uncharacterized. Using bimolecular fluorescence complementation (BiFC), we found that all four ER‐localized Arabidopsis Cb5 isoforms (AtCb5–B, ‐C, ‐D and ‐E) interact with RTE1 in plant cells. In support of this interaction, atcb5 mutants exhibited phenotypic parallels with rte1 mutants in Arabidopsis. Phenotypes included partial suppression of etr1–2 ethylene insensitivity, and no suppression of RTE1‐independent ethylene receptor isoforms. The single loss‐of‐function mutants atcb5–b, ‐c and ‐d appeared similar to the wild‐type, but double mutant combinations displayed slight ethylene hypersensitivity. Over‐expression of AtCb5–D conferred reduced ethylene sensitivity similar to that conferred by RTE1 over‐expression, and genetic analyses suggested that AtCb5–D acts upstream of RTE1 in the ethylene response. These findings suggest an unexpected role for Cb5, in which Cb5 and RTE1 are functional partners in promoting ETR1‐mediated repression of ethylene signaling.     

Arabidopsis CPR5 regulates ethylene signaling via molecular association with the ETR1 receptor

The plant hormone ethylene plays various functions in plant growth, development and response to environmental stress. Ethylene is perceived by membrane‐bound ethylene receptors, and among the homologous receptors in Arabidopsis, the ETR1 ethylene receptor plays a major role. The present study provides evidence demonstrating that Arabidopsis CPR5 functions as a novel ETR1 receptor‐interacting protein in regulating ethylene response and signaling. Yeast split ubiquitin assays and bi‐fluorescence complementation studies in plant cells indicated that CPR5 directly interacts with the ETR1 receptor. Genetic analyses indicated that mutant alleles of cpr5 can suppress ethylene insensitivity in both etr1‐1 and etr1‐2, but not in other dominant ethylene receptor mutants. Overexpression of Arabidopsis CPR5 either in transgenic Arabidopsis plants, or ectopically in tobacco, significantly enhanced ethylene sensitivity. These findings indicate that CPR5 plays a critical role in regulating ethylene signaling. CPR5 is localized to endomembrane structures and the nucleus, and is involved in various regulatory pathways, including pathogenesis, leaf senescence, and spontaneous cell death. This study provides evidence for a novel regulatory function played by CPR5 in the ethylene receptor signaling pathway in Arabidopsis.     

Reduced L‐arginine transport contributes to the pathogenesis of myocardial ischemia‐reperfusion injury

Myocardial injury due to ischemia‐reperfusion (I‐R) damage remains a major clinical challenge. Its pathogenesis is complex including endothelial dysfunction and heightened oxidative stress although the key driving mechanism remains uncertain. In this study we tested the hypothesis that the I‐R process induces a state of insufficient L ‐arginine availability for NO biosynthesis, and that this is pivotal in the development of myocardial I‐R damage. In neonatal rat ventricular cardiomyocytes (NVCM), hypoxia‐reoxygenation significantly decreased L ‐arginine uptake and NO production (42 ± 2% and 71 ± 4%, respectively, both P < 0.01), maximal after 2 h reoxygenation. In parallel, mitochondrial membrane potential significantly decreased and ROS production increased (both P < 0.01). NVCMs infected with adenovirus expressing the L ‐arginine transporter, CAT1, and NVCMs supplemented with L ‐arginine both exhibited significant (all P < 0.05) improvements in NO generation and mitochondrial membrane potentials, with a concomitant significant fall in ROS production and lactate dehydrogenase release during hypoxia‐reoxygenation. In contrast, L ‐arginine deprived NVCM had significantly worsened responses to hypoxia‐reoxygenation. In isolated perfused mouse hearts, L ‐arginine infusion during reperfusion significantly improved left ventricular function after I‐R. These improved contractile responses were not dependent on coronary flow but were associated with a significant decrease in nitrotyrosine formation and increases in phosphorylation of both Akt and troponin I. Collectively, these data strongly implicate reduced L ‐arginine availability as a key factor in the pathogenesis of I‐R injury. Increasing L ‐arginine availability via increased CAT1 expression or by supplementation improves myocardial responses to I‐R. Restoration of L ‐arginine availability may therefore be a valuable strategy to ameliorate I‐R injury. J. Cell. Biochem. 108: 156–168, 2009. © 2009 Wiley‐Liss, Inc.     

Instability of BLOC‐2 and BLOC‐3 in Chinese patients with Hermansky‐Pudlak syndrome

Hermansky‐Pudlak syndrome (HPS) is a rare recessive disorder characterized by oculocutaneous albinism (OCA) or ocular albinism (OA), bleeding tendency, and other symptoms due to multiple defects in tissue‐specific lysosome‐related organelles. Ten HPS subtypes have been characterized with mutations in HPS1 to HPS10, which encode the subunits of BLOC‐1, ‐2, ‐3, and AP‐3. Using next‐generation sequencing (NGS), we have screened 100 hypopigmentation genes in OCA or OA patients and identified four HPS‐1, one HPS‐3, one HPS‐4, one HPS‐5, and three HPS‐6. The HPS‐4 case is the first report in the Chinese population. Among these 20 mutational alleles, 16 were previously unreported alleles (6 in HPS1, 1 in HPS3, 2 in HPS4, 2 in HPS5, and 5 in HPS6). BLOC‐2 and BLOC‐3 were destabilized due to the mutation of these HPS genes which are so far the only reported causative genes in Chinese HPS patients, in which HPS‐1 and HPS‐6 are the most common subtypes. The mutational spectrum of Chinese HPS is population specific.     

Nonaqueous fractionation and overexpression of fluorescent‐tagged enzymes reveals the subcellular sites of L‐theanine biosynthesis in tea

l ‐Theanine is a specialized metabolite in the tea (Camellia sinensis) plant which can constitute over 50% of the total amino acids. This makes an important contribution to tea functionality and quality, but the subcellular location and mechanism of biosynthesis of l ‐theanine are unclear. Here, we identified five distinct genes potentially capable of synthesizing l ‐theanine in tea. Using a nonaqueous fractionation method, we determined the subcellular distribution of l ‐theanine in tea shoots and roots and used transient expression in Nicotiana or Arabidopsis to investigate in vivo functions of l ‐theanine synthetase and also to determine the subcellular localization of fluorescent‐tagged proteins by confocal laser scanning microscopy. In tea root tissue, the cytosol was the main site of l ‐theanine biosynthesis, and cytosol‐located CsTSI was the key l ‐theanine synthase. In tea shoot tissue, l ‐theanine biosynthesis occurred mainly in the cytosol and chloroplasts and CsGS1.1 and CsGS2 were most likely the key l ‐theanine synthases. In addition, l ‐theanine content and distribution were affected by light in leaf tissue. These results enhance our knowledge of biochemistry and molecular biology of the biosynthesis of functional tea compounds.     

Influence of Mannosylation on Immunostimulating Activity of Adamant‐1‐yl Tripeptide

The mannosylated derivative of adamant‐1‐yl tripeptide (D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) was prepared to study the effects of mannosylation on adjuvant (immunostimulating) activity. Mannosylated adamant‐1‐yl tripeptide (Man‐OCH₂CH(Me)CO‐D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) is a non‐pyrogenic, H₂O‐soluble, and non‐toxic compound. Adjuvant activity of mannosylated adamantyl tripeptide was tested in the mouse model with ovalbumin as an antigen and in comparison to the parent tripeptide and peptidoglycan monomer (PGM, β‐D ‐GlcNAc‐(1→4)‐D ‐MurNAc‐L ‐Ala‐D ‐isoGln‐mesoDAP(εNH₂)‐D ‐Ala‐D ‐Ala), a well‐known effective adjuvant. The mannosylation of adamantyl tripeptide caused the amplification of its immunostimulating activity in such a way that it was comparable to that of PGM.     

Chromatographic profiles of tryptophan and kynurenine enantiomers derivatized with (S)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole using LC–MS/MS on a triazole‐bonded column

d ‐ and l ‐Tryptophan (Trp) and d ‐ and l ‐kynurenine (KYN) were derivatized with a chiral reagent, (S )‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐PyNCS), and were separated enantiomerically by high‐performance liquid chromatography (HPLC) equipped with a triazole‐bonded column (Cosmosil HILIC) using tandem mass spectrometric (MS/MS) detection. Effects of column temperature, salt (HCO₂NH₄) concentration, and pH of the mobile phase in the enantiomeric separation, followed by MS detection of (S )‐DBD‐PyNCS‐d ,l ‐Trp and ‐d ,l ‐KYN, were investigated. The mobile phase consisting of CH₃CN/10 mM ammonium formate in H₂O (pH 5.0) (90/10) with a column temperature of 50–60 °C gave satisfactory resolution (R s) and mass‐spectrometric detection. The enantiomeric separation of d ,l ‐Trp and d ,l ‐KYN produced R s values of 2.22 and 2.13, and separation factors (α) of 1.08 and 1.08, for the Trp and KYN enantiomers, respectively. The proposed LC–MS/MS method provided excellent detection sensitivity of both enantiomers of Trp and KYN (5.1–19 nM).     

Cyclo(d‐Tyr‐d‐Phe): a new antibacterial,anticancer, and antioxidant cyclic dipeptide from Bacillus sp. N strain associated with a rhabditid entomopathogenic nematode

A new microbial cyclic dipeptide (diketopiperazine), cyclo(d ‐Tyr‐d ‐Phe) was isolated for the first time from the ethyl acetate extract of fermented modified nutrient broth of Bacillus sp. N strain associated with rhabditid Entomopathogenic nematode. Antibacterial activity of the compound was determined by minimum inhibitory concentration and agar disc diffusion method against medically important bacteria and the compound recorded significant antibacterial against test bacteria. Highest activity was recorded against Staphylococcus epidermis (1 µg/ml) followed by Proteus mirabilis (2 µg/ml). The activity of cyclo(d ‐Tyr‐d ‐Phe) against S. epidermis is better than chloramphenicol, the standard antibiotics. Cyclo(d ‐Tyr‐d ‐Phe) recorded significant antitumor activity against A549 cells (IC₅₀ value: 10 μM) and this compound recorded no cytotoxicity against factor signaling normal fibroblast cells up to 100 μM. Cyclo(d ‐Tyr‐d ‐Phe) induced significant morphological changes and DNA fragmentation associated with apoptosis in A549 cells. Acridine orange/ethidium bromide stained cells indicated apoptosis induction by cyclo(d ‐Tyr‐d ‐Phe). Flow cytometry analysis showed that the cyclo(d ‐Tyr‐d ‐Phe) did not induce cell cycle arrest. Effector molecule of apoptosis such as caspase‐3 was found activated in treated cells, suggesting apoptosis as the main mode of cell death. Antioxidant activity was evaluated by free radical scavenging and reducing power activity, and the compound recorded significant antioxidant activity. The free radical scavenging activity of cyclo(d ‐Tyr‐d ‐Phe) is almost equal to that of butylated hydroxyanisole, the standard antioxidant agent. We also compared the biological activity of natural cyclo(d ‐Tyr‐d ‐Phe) with synthetic cyclo(d ‐Tyr‐d ‐Phe) and cyclo(l ‐Tyr‐l ‐Phe). Natural and synthetic cyclo(d ‐Tyr‐d ‐Phe) recorded similar pattern of activity. Although synthetic cyclo(l ‐Tyr‐l ‐Phe) recorded lower activity. But in the case of reducing power activity, synthetic cyclo(l ‐Tyr‐l ‐Phe) recorded significant activity than natural and synthetic cyclo(d ‐Tyr‐d ‐Phe). The results of the present study reveals that cyclo(d ‐Tyr‐d ‐Phe) is more bioactive than cyclo(l ‐Tyr‐l ‐Phe). To the best of our knowledge, this is the first time that cyclo(d ‐Tyr‐d ‐Phe) has been isolated from microbial natural source and also the antibacterial, anticancer, and antioxidant activity of cyclo(d ‐Tyr‐d ‐Phe) is also reported for the first time. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Ethylene‐dependent aerenchyma formation in adventitious roots is regulated differently in rice and maize

In roots of gramineous plants, lysigenous aerenchyma is created by the death and lysis of cortical cells. Rice (Oryza sativa) constitutively forms aerenchyma under aerobic conditions, and its formation is further induced under oxygen‐deficient conditions. However, maize (Zea mays) develops aerenchyma only under oxygen‐deficient conditions. Ethylene is involved in lysigenous aerenchyma formation. Here, we investigated how ethylene‐dependent aerenchyma formation is differently regulated between rice and maize. For this purpose, in rice, we used the reduced culm number1 (rcn1) mutant, in which ethylene biosynthesis is suppressed. Ethylene is converted from 1‐aminocyclopropane‐1‐carboxylic acid (ACC) by the action of ACC oxidase (ACO). We found that OsACO5 was highly expressed in the wild type, but not in rcn1, under aerobic conditions, suggesting that OsACO5 contributes to aerenchyma formation in aerated rice roots. By contrast, the ACO genes in maize roots were weakly expressed under aerobic conditions, and thus ACC treatment did not effectively induce ethylene production or aerenchyma formation, unlike in rice. Aerenchyma formation in rice roots after the initiation of oxygen‐deficient conditions was faster and greater than that in maize. These results suggest that the difference in aerenchyma formation in rice and maize is due to their different mechanisms for regulating ethylene biosynthesis.