The role of root apoplastic transport barriers in salt tolerance of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Increasing soil salinity reduces crop yields worldwide, with rice being particularly affected. We have examined the correlation between apoplastic barrier formation in roots, Na⁺ uptake into shoots and plant survival for three rice (Oryza sativa L.) cultivars of varying salt sensitivity: the salt-tolerant Pokkali, moderately tolerant Jaya and sensitive IR20. Rice plants grown hydroponically or in soil for 1 month were subjected to both severe and moderate salinity stress. Apoplastic barriers in roots were visualized using fluorescence microscopy and their chemical composition determined by gas chromatography and mass spectrometry. Na⁺ content was estimated by flame photometry. Suberization of apoplastic barriers in roots of Pokkali was the most extensive of the three cultivars, while Na⁺ accumulation in the shoots was the least. Saline stress induced the strengthening of these barriers in both sensitive and tolerant cultivars, with increase in mRNAs encoding suberin biosynthetic enzymes being detectable within 30 min of stress. Enhanced barriers were detected after several days of moderate stress. Overall, more extensive apoplastic barriers in roots correlated with reduced Na⁺ uptake and enhanced survival when challenged with high salinity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Flux balance analysis of barley seeds: a computational approach to study systemic properties of central metabolism

The accumulation of storage compounds is an important aspect of cereal seed metabolism. Due to the agronomical importance of the storage reserves of starch, protein, and oil, the understanding of storage metabolism is of scientific interest, with practical applications in agronomy and plant breeding. To get insight into storage patterning in developing cereal seed in response to environmental and genetic perturbation, a computational analysis of seed metabolism was performed. A metabolic network of primary metabolism in the developing endosperm of barley (Hordeum vulgare), a model plant for temperate cereals, was constructed that includes 257 biochemical and transport reactions across four different compartments. The model was subjected to flux balance analysis to study grain yield and metabolic flux distributions in response to oxygen depletion and enzyme deletion. In general, the simulation results were found to be in good agreement with the main biochemical properties of barley seed storage metabolism. The predicted growth rate and the active metabolic pathway patterns under anoxic, hypoxic, and aerobic conditions predicted by the model were in accordance with published experimental results. In addition, the model predictions gave insight into the potential role of inorganic pyrophosphate metabolism to maintain seed metabolism under oxygen deprivation.     

Loss of TMEM16A Causes a Defect in Epithelial Ca2+-dependent Chloride Transport

Molecular identification of the Ca²⁺-dependent chloride channel TMEM16A (ANO1) provided a fundamental step in understanding Ca²⁺-dependent Cl⁻ secretion in epithelia. TMEM16A is an intrinsic constituent of Ca²⁺-dependent Cl⁻ channels in cultured epithelia and may control salivary output, but its physiological role in native epithelial tissues remains largely obscure. Here, we demonstrate that Cl⁻ secretion in native epithelia activated by Ca²⁺-dependent agonists is missing in mice lacking expression of TMEM16A. Ca²⁺-dependent Cl⁻ transport was missing or largely reduced in isolated tracheal and colonic epithelia, as well as hepatocytes and acinar cells from pancreatic and submandibular glands of TMEM16A^−/− animals. Measurement of particle transport on the surface of tracheas ex vivo indicated largely reduced mucociliary clearance in TMEM16A^−/− mice. These results clearly demonstrate the broad physiological role of TMEM16A^−/− for Ca²⁺-dependent Cl⁻ secretion and provide the basis for novel treatments in cystic fibrosis, infectious diarrhea, and Sjöegren syndrome.Electrolyte secretion in epithelial tissues is based on the major second messenger pathways cAMP and Ca²⁺, which activate the cystic fibrosis transmembrane conductance regulator (CFTR)² Cl⁻ channels and Ca²⁺-dependent Cl⁻ channels, respectively (1–3). CFTR conducts Cl⁻ in epithelial cells of airways, intestine, and the ducts of pancreas and sweat gland, while Ca²⁺-dependent Cl⁻ channels secrete Cl⁻ in pancreatic acini and salivary and sweat glands (4–6). Controversy exists as to the contribution of these channels to Cl⁻ secretion in submucosal glands of airways and the relevance for cystic fibrosis (7–9). While cAMP-dependent Cl⁻ secretion by CFTR is well examined, detailed analysis of epithelial Ca²⁺-dependent Cl⁻ secretion is hampered by the lack of a molecular counterpart. Although bestrophins may form Ca²⁺-dependent Cl⁻ channels and facilitate Ca²⁺-dependent Cl⁻ secretion in epithelial tissues (10, 11), they are unlikely to form secretory Cl⁻ channels in the apical cell membrane, because Ca²⁺-dependent Cl⁻ secretion is still present in epithelia of mice lacking expression of bestrophin (12). Bestrophins may rather have an intracellular function by facilitating receptor mediated Ca²⁺ signaling and activation of membrane localized channels (13). With the discovery that TMEM16A produces Ca²⁺-activated Cl⁻ currents with biophysical and pharmacological properties close to those in native epithelial tissues, these proteins are now very likely candidates for endogenous Ca²⁺-dependent Cl⁻ channels (14–17). In cultured airway epithelial cells, small interfering RNA knockdown of endogenous TMEM16A largely reduced calcium-dependent chloride secretion (16). However, apart from preliminary studies of airways and salivary glands, the physiological significance of TMEM16A in native epithelia, particularly in glands, is unclear (14, 17).     

TMEM16 Proteins Produce Volume-regulated Chloride Currents That Are Reduced in Mice Lacking TMEM16A

All vertebrate cells regulate their cell volume by activating chloride channels of unknown molecular identity, thereby activating regulatory volume decrease. We show that the Ca²⁺-activated Cl⁻ channel TMEM16A together with other TMEM16 proteins are activated by cell swelling through an autocrine mechanism that involves ATP release and binding to purinergic P2Y₂ receptors. TMEM16A channels are activated by ATP through an increase in intracellular Ca²⁺ and a Ca²⁺-independent mechanism engaging extracellular-regulated protein kinases (ERK1/2). The ability of epithelial cells to activate a Cl⁻ conductance upon cell swelling, and to decrease their cell volume (regulatory volume decrease) was dependent on TMEM16 proteins. Activation of I_Cl,swell was reduced in the colonic epithelium and in salivary acinar cells from mice lacking expression of TMEM16A. Thus TMEM16 proteins appear to be a crucial component of epithelial volume-regulated Cl⁻ channels and may also have a function during proliferation and apoptotic cell death.Regulation of cell volume is fundamental to all cells, particularly during cell growth and division. External hypotonicity leads to cell swelling and subsequent activation of volume-regulated chloride and potassium channels, to release intracellular ions and to re-shrink the cells, a process termed regulatory volume decrease (RVD)³ (1). Volume-regulated chloride currents (I_Cl,swell) have dual functions during cell proliferation as well as apoptotic volume decrease (AVD), preceding apoptotic cell death (2). Although I_Cl,swell is activated in swollen cells to induce RVD, AVD takes place under normotonic conditions to shrink cells (3, 4). Early work suggested intracellular Ca²⁺ as an important mediator for activation of I_Cl,swell and volume-regulated K⁺ channels (5), whereas subsequent studies only found a permissive role of Ca²⁺ for activation of I_Cl,swell (6), reviewed in Ref. 1. In addition, a plethora of factors and signaling pathways have been implicated in activation of I_Cl,swell, making cell volume regulation an extremely complex process (reviewed in Refs. 1, 3, and 7). These factors include intracellular ATP, the cytoskeleton, phospholipase A2-dependent pathways, and protein kinases such as extracellular-regulated kinase ERK1/2 (reviewed in Refs. 1 and 7). Previous approaches in identifying swelling-activated Cl⁻ channels have been unsuccessful or have produced controversial data. Thus none of the previous candidates such as pI_Cln, the multidrug resistance protein, or ClC-3 are generally accepted to operate as volume-regulated Cl⁻ channels (reviewed in Refs. 8 and 9). Notably, the cystic fibrosis transmembrane conductance regulator (CFTR) had been shown in earlier studies to influence I_Cl,swell and volume regulation (10–12). The variable properties of I_Cl,swell suggest that several gene products may affect I_Cl,swell in different cell types.The TMEM16 transmembrane protein family consists of 10 different proteins with numerous splice variants that contain 8–9 transmembrane domains and have predicted intracellular N- and C-terminal tails (13, 16–18). TMEM16A (also called ANO1) is required for normal development of the murine trachea (14) and is associated with different types of tumors, dysplasia, and nonsyndromic hearing impairment (13, 15). TMEM16A has been identified as a subunit of Ca²⁺-activated Cl⁻ channels that are expressed in epithelial and non-epithelial tissues (16–18). Interestingly, members of the TMEM16 family have been suggested to play a role in osmotolerance in Saccharomyces cerevisiae (19). Here we show that TMEM16 proteins also contribute to I_Cl,swell and regulatory volume decrease.     

Crystal structure of the human tRNA microhelix isoacceptor G9990 at 1.18 Å resolution

The tRNA^Gly/Glycyl-tRNA synthetase system belongs to the so called ‘class II’ in which tRNA identity elements consist of relative few and simple motifs, as compared to ‘class I’ where the tRNA determinants are more complicated and spread over different parts of the tRNA, mostly including the anticodon. The determinants from ‘class II’ although, are located in the aminoacyl stem and sometimes include the discriminator base. There exist predominant structure differences for the Glycyl-tRNA-synthetases and for the tRNA^Gly identity elements comparing eucaryotic/archaebacterial and eubacterial systems.We focus on comparative X-ray structure analysis of tRNA^Gly acceptor stem microhelices from different organisms. Here, we report the X-ray structure of the human tRNA^Gly microhelix isoacceptor G9990 at 1.18 Å resolution. Superposition experiments to another human tRNA^Gly microhelix and a detailed comparison of the RNA hydration patterns show a great number of water molecules with identical positions in both RNAs. This is the first structure comparison of hydration layers from two isoacceptor tRNA microhelices with a naturally occurring base pair exchange.     

AgCl precipitates in isolated cuticular membranes reduce rates of cuticular transpiration

Counter diffusion of chloride, applied as NaCl at the inner side of isolated cuticles, and silver, applied as AgNO₃ at the outer side, lead to the formation of insoluble AgCl precipitates in isolated cuticles. AgCl precipitates could be visualized by light and scanning electron microscopy. The presence of AgCl precipitates in isolated cuticles was verified by energy dispersive X-ray analysis. It is argued that insoluble AgCl precipitates formed in polar pores of cuticles and as a consequence, cuticular transpiration of 13 out of 15 investigated species was significantly reduced up to three-fold. Water as a small and uncharged but polar molecule penetrates cuticles via two parallel paths: a lipophilic path, formed by lipophilic cutin and wax domains, and a aqueous pathe, formed by polar pores. Thus, permeances P (m s⁻¹) of water, which is composed of the two quantities P _Lipid and P _Pore, decreased, since water transport across polar pores was affected by AgCl precipitates. Cuticles with initially high rates of cuticular transpiration were generally more sensitive towards AgCl precipitates compared to cuticles with initially low rates of transpiration. Results presented here, significantly improves the current model of the structure of the cuticular transpiration barrier, since the pronounced heterogeneity of the cuticular transport barrier, composed of lipophilic as well as polar paths of diffusion, has to be taken into account in future.     

PARP-2 deficiency affects the survival of CD4+CD8+ double-positive thymocytes

Poly-(ADP-ribose) polymerase-2 (PARP-2) belongs to a large family of enzymes that synthesize and transfer ADP-ribose polymers to acceptor proteins, modifying their functional properties. PARP-2-deficient (Parp-2-/-) cells, similar to Parp-1-/- cells, are sensitive to both ionizing radiation and alkylating agents. Here we show that inactivation of mouse Parp-2, but not Parp-1, produced a two-fold reduction in CD4+CD8+ double-positive (DP) thymocytes associated with decreased DP cell survival. Microarray analyses revealed increased expression of the proapoptotic Bcl-2 family member Noxa in Parp-2-/- DP thymocytes compared to littermate controls. In addition, DP thymocytes from Parp-2-/- have a reduced expression of T-cell receptor (TCR)alpha and a skewed repertoire of TCRalpha toward the 5' Jalpha segments. Our results show that in the absence of PARP-2, the survival of DP thymocytes undergoing TCRalpha recombination is compromised despite normal amounts of Bcl-xL. These data suggest a novel role for PARP-2 as an important mediator of T-cell survival during thymopoiesis by preventing the activation of DNA damage-dependent apoptotic response during the multiple rounds of TCRalpha rearrangements preceding a positively selected TCR.     

Influence of polymorphisms in the NOD1/CARD4 and NOD2/CARD15 genes on the clinical outcome of Helicobacter pylori infection

Host immune response influences the clinical outcome of Helicobacter pylori infection leading to ulcer disease, gastric carcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. A genetic risk profile for gastric cancer has been identified, but genetic susceptibility to develop MALT lymphoma is still unclear. We investigated the role of NOD1 and NOD2 as intracellular recognition molecules for pathogen-associated molecules in H. pylori infection in vitro and analysed the influence of single nucleotide polymorphisms on susceptibility to ulcer disease and MALT lymphoma. Expression of NOD1 and NOD2 significantly sensitized HEK293 cells to H. pylori-induced NF-kappaB activation in a cag pathogenicity island (cagPAI)-dependent manner. In cells carrying the Crohn-associated NOD2 variant R702W the NF-kappaB response was significantly diminished. NOD1/NOD2 expression levels were induced in the gastric epithelium in H. pylori-positive patients. No mutations were found to be associated with gastritis or gastric ulcer development. However, the R702W mutation in the NOD2/CARD15 gene was significantly associated with gastric lymphoma. Carrier of the rare allele T had a more than doubled risk to develop lymphoma than controls [odds ratio (OR): 2.4, 95% confidence interval (CI): 1.2-4.6; P < 0.044]. H. pylori-induced upregulation of NOD1 and NOD2 in vivo may play a critical role in the recognition of this common pathogen. A missense mutation in the leucine-rich region of CARD15 is associated with gastric lymphoma.     

PARP1 Is a TRF2-associated poly(ADP-ribose)polymerase and protects eroded telomeres

Poly(ADP-ribose)polymerase 1 (PARP1) is well characterized for its role in base excision repair (BER), where it is activated by and binds to DNA breaks and catalyzes the poly(ADP-ribosyl)ation of several substrates involved in DNA damage repair. Here we demonstrate that PARP1 associates with telomere repeat binding factor 2 (TRF2) and is capable of poly(ADP-ribosyl)ation of TRF2, which affects binding of TRF2 to telomeric DNA. Immunostaining of interphase cells or metaphase spreads shows that PARP1 is detected sporadically at normal telomeres, but it appears preferentially at eroded telomeres caused by telomerase deficiency or damaged telomeres induced by DNA-damaging reagents. Although PARP1 is dispensable in the capping of normal telomeres, Parp1 deficiency leads to an increase in chromosome end-to-end fusions or chromosome ends without detectable telomeric DNA in primary murine cells after induction of DNA damage. Our results suggest that upon DNA damage, PARP1 is recruited to damaged telomeres, where it can help protect telomeres against chromosome end-to-end fusions and genomic instability.