Genotypic and tissue-specific variation of Populus nigra transcriptome profiles in response to drought.

Climate change is one of the most important challenges for mankind in the far and near future. In this regard, sustainable production of woody crops on marginal land with low water availability is a major challenge to tackle. This dataset is part of an experiment, in which we exposed three genetically differentiated genotypes of Populus nigra originating from contrasting natural habitats to gradually increasing moderate drought. RNA sequencing was performed on fine roots, developing xylem and leaves of those three genotypes under control and moderate drought conditions in order to get a comprehensive dataset on the transcriptional changes at the whole plant level under water limiting conditions. This dataset has already provided insight in the transcriptional control of saccharification potential of the three Populus genotypes under drought conditions and we suggest that our data will be valuable for further in-depth analysis regarding candidate gene identification or, on a bigger scale, for meta-transcriptome analysis.

The influence of transpiration on foliar accumulation of salt and nutrients under salinity in poplar (Populus × canescens).

Increasing salinity is one of the major drawbacks for plant growth. Besides the ion itself being toxic to plant cells, it greatly interferes with the supply of other macronutrients like potassium, calcium and magnesium. However, little is known about how sodium affects the translocation of these nutrients from the root to the shoot. The major driving force of this translocation process is thought to be the water flow through the xylem driven by transpiration. To dissect the effects of transpiration from those of salinity we compared salt stressed, ABA treated and combined salt- and ABA treated poplars with untreated controls. Salinity reduced the root content of major nutrients like K+, Ca2+ and Mg2+. Less Ca2+ and Mg2+ in the roots resulted in reduced leaf Ca2+ and leaf Mg2+ levels due to reduced stomatal conductance and reduced transpiration. Interestingly, leaf K+ levels were positively affected in leaves under salt stress although there was less K+ in the roots under salt. In response to ABA, transpiration was also decreased and Mg2+ and Ca2+ levels decreased comparably to the salt stress treatment, while K+ levels were not affected. Thus, our results suggest that loading and retention of leaf K+ is enhanced under salt stress compared to merely transpiration driven cation supply.

Engineering Drought Resistance in Forest Trees.

Climatic stresses limit plant growth and productivity. In the past decade, tree improvement programs were mainly focused on yield but it is obvious that enhanced stress resistance is also required. In this review we highlight important drought avoidance and tolerance mechanisms in forest trees. Genomes of economically important trees species with divergent resistance mechanisms can now be exploited to uncover the mechanistic basis of long-term drought adaptation at the whole plant level. Molecular tree physiology indicates that osmotic adjustment, antioxidative defense and increased water use efficiency are important targets for enhanced drought tolerance at the cellular and tissue level. Recent biotechnological approaches focused on overexpression of genes involved in stress sensing and signaling, such as the abscisic acid core pathway, and down-stream transcription factors. By this strategy, a suite of defense systems was recruited, generally enhancing drought and salt stress tolerance under laboratory conditions. However, field studies are still scarce. Under field conditions trees are exposed to combinations of stresses that vary in duration and magnitude. Variable stresses may overrule the positive effect achieved by engineering an individual defense pathway. To assess the usability of distinct modifications, large-scale experimental field studies in different environments are necessary. To optimize the balance between growth and defense, the use of stress-inducible promoters may be useful. Future improvement programs for drought resistance will benefit from a better understanding of the intricate networks that ameliorate molecular and ecological traits of forest trees.

The vacuolar calcium sensors CBL2 and CBL3 affect seed size and embryonic development in Arabidopsis thaliana.

Stimulus-specific calcium (Ca(2+) ) signals have crucial functions in developmental processes in many organisms, and are deciphered by various Ca(2+) -binding proteins. In Arabidopsis thaliana, a signaling network consisting of calcineurin B-like (CBL) protein calcium sensors and CBL-interacting protein kinases (CIPKs) has been shown to fulfil pivotal functions at the plasma membrane in regulating ion fluxes and abiotic stress responses. However, the role of tonoplast-localized CBL proteins and especially their function in regulating developmental programs remains largely unknown. In this study, we analyzed single and double mutants of the closely related tonoplast-localized calcium sensors CBL2 and CBL3, which show either reduction of function (rf) or complete loss of function (lf). While single cbl2 or cbl3 mutants did not display discernable phenotypes, cbl2/cbl3 mutants exhibited defects in vegetative growth and were severely impaired in seed development and morphology. Seeds of the cbl2/3rf mutant were smaller in size and exhibited reduced weight and fatty acid content compared to wild-type, but accumulation of sucrose was not altered. Moreover, accumulation of inositol hexakisphosphate (InsP6 ), the major storage form of phosphorus in seeds, was significantly reduced in mutant seeds. In addition, complete loss of CBL2 and CBL3 function in cbl2/3lf resulted in a high frequency of severe defects in embryonic development. Together, our findings reveal a crucial function of Ca(2+) -controlled processes at the vacuolar membrane as determinants of seed yield and size, and demonstrate the importance of vacuolar CBL calcium sensors for plant embryogenesis.

Histone deacetylase 10 promotes autophagy-mediated cell survival.

Tumor cells activate autophagy in response to chemotherapy-induced DNA damage as a survival program to cope with metabolic stress. Here, we provide in vitro and in vivo evidence that histone deacetylase (HDAC)10 promotes autophagy-mediated survival in neuroblastoma cells. We show that both knockdown and inhibition of HDAC10 effectively disrupted autophagy associated with sensitization to cytotoxic drug treatment in a panel of highly malignant V-MYC myelocytomatosis viral-related oncogene, neuroblastoma derived-amplified neuroblastoma cell lines, in contrast to nontransformed cells. HDAC10 depletion in neuroblastoma cells interrupted autophagic flux and induced accumulation of autophagosomes, lysosomes, and a prominent substrate of the autophagic degradation pathway, p62/sequestosome 1. Enforced HDAC10 expression protected neuroblastoma cells against doxorubicin treatment through interaction with heat shock protein 70 family proteins, causing their deacetylation. Conversely, heat shock protein 70/heat shock cognate 70 was acetylated in HDAC10-depleted cells. HDAC10 expression levels in high-risk neuroblastomas correlated with autophagy in gene-set analysis and predicted treatment success in patients with advanced stage 4 neuroblastomas. Our results demonstrate that HDAC10 protects cancer cells from cytotoxic agents by mediating autophagy and identify this HDAC isozyme as a druggable regulator of advanced-stage tumor cell survival. Moreover, these results propose a promising way to considerably improve treatment response in the neuroblastoma patient subgroup with the poorest outcome.

A new ß-estradiol-inducible vector set that facilitates easy construction and efficient expression of transgenes reveals CBL3-dependent cytoplasm to tonoplast translocation of CIPK5.

Transient and stable expression of transgenes is central to many investigations in plant biology research. Chemical regulation of expression can circumvent problems of plant lethality caused by constitutive overexpression or allow inducible knock (out/down) approaches. Several chemically inducible or repressible systems have been described and successfully applied. However, cloning and application-specific modification of most available inducible expression systems have been limited and remained complicated due to restricted cloning options. Here we describe a new set of 57 vectors that enable transgene expression in transiently or stably transformed cells. All vectors harbor a synthetically optimized XVE expression cassette, allowing ß-estradiol mediated protein expression. Plasmids are equipped with the reporter genes GUS, GFP, mCherry, or with HA and StrepII epitope tags and harbor an optimized multiple cloning site for flexible and simple cloning strategies. Moreover, the vector design allows simple substitution of the driving promoter to achieve tissue-specificity or to modulate expression ranges of inducible transgene expression. We report details of the kinetics and dose-dependence of expression induction in Arabidopsis leaf mesophyll protoplasts, transiently transformed Nicotiana benthamiana leaves, and stably transformed Arabidopsis plants. Using these vectors, we investigated the influence of CBL (Calcineurin B-like) protein expression on the subcellular localization of CIPKs (Calcineurin B-like interacting protein kinases). These analyses uncovered that induced co-expression of CBL3 is fully sufficient for dynamic translocation of CIPK5 from the cytoplasm to the tonoplast. Thus, the vector system presented here facilitates a broad range of research applications.

Conservation and divergence between cytoplasmic and muscle-specific actin capping proteins: insights from the crystal structure of cytoplasmic Cap32/34 from Dictyostelium discoideum.

BACKGROUND: Capping protein (CP), also known as CapZ in muscle cells and Cap32/34 in Dictyostelium discoideum, plays a major role in regulating actin filament dynamics. CP is a ubiquitously expressed heterodimer comprising an α- and ß-subunit. It tightly binds to the fast growing end of actin filaments, thereby functioning as a "cap" by blocking the addition and loss of actin subunits. Vertebrates contain two somatic variants of CP, one being primarily found at the cell periphery of non-muscle tissues while the other is mainly localized at the Z-discs of skeletal muscles. RESULTS: To elucidate structural and functional differences between cytoplasmic and sarcomercic CP variants, we have solved the atomic structure of Cap32/34 (32=ß- and 34=α-subunit) from the cellular slime mold Dictyostelium at 2.2 Å resolution and compared it to that of chicken muscle CapZ. The two homologs display a similar overall arrangement including the attached α-subunit C-terminus (α-tentacle) and the flexible ß-tentacle. Nevertheless, the structures exhibit marked differences suggesting considerable structural flexibility within the α-subunit. In the α-subunit we observed a bending motion of the ß-sheet region located opposite to the position of the C-terminal ß-tentacle towards the antiparallel helices that interconnect the heterodimer. Recently, a two domain twisting attributed mainly to the ß-subunit has been reported. At the hinge of these two domains Cap32/34 contains an elongated and highly flexible loop, which has been reported to be important for the interaction of cytoplasmic CP with actin and might contribute to the more dynamic actin-binding of cytoplasmic compared to sarcomeric CP (CapZ). CONCLUSIONS: The structure of Cap32/34 from Dictyostelium discoideum allowed a detailed analysis and comparison between the cytoplasmic and sarcomeric variants of CP. Significant structural flexibility could particularly be found within the α-subunit, a loop region in the ß-subunit, and the surface of the α-globule where the amino acid differences between the cytoplasmic and sarcomeric mammalian CP are located. Hence, the crystal structure of Cap32/34 raises the possibility of different binding behaviours of the CP variants toward the barbed end of actin filaments, a feature, which might have arisen from adaptation to different environments.

Phosphorylation of calcineurin B-like (CBL) calcium sensor proteins by their CBL-interacting protein kinases (CIPKs) is required for full activity of CBL-CIPK complexes toward their target proteins.

Calcineurin B-like proteins (CBLs) represent a family of calcium sensor proteins that interact with a group of serine/threonine kinases designated as CBL-interacting protein kinases (CIPKs). CBL-CIPK complexes are crucially involved in relaying plant responses to many environmental signals and in regulating ion fluxes. However, the biochemical characterization of CBL-CIPK complexes has so far been hampered by low activities of recombinant CIPKs. Here, we report on an efficient wheat germ extract-based in vitro transcription/translation protocol that yields active full-length wild-type CIPK proteins. We identified a conserved serine residue within the C terminus of CBLs as being phosphorylated by their interacting CIPKs. Remarkably, our studies revealed that CIPK-dependent CBL phosphorylation is strictly dependent on CBL-CIPK interaction via the CIPK NAF domain. The phosphorylation status of CBLs does not appear to influence the stability, localization, or CIPK interaction of these calcium sensor proteins in general. However, proper phosphorylation of CBL1 is absolutely required for the in vivo activation of the AKT1 K(+) channel by CBL1-CIPK23 and CBL9-CIPK23 complexes in oocytes. Moreover, we show that by combining CBL1, CIPK23, and AKT1, we can faithfully reconstitute CBL-dependent enhancement of phosphorylation of target proteins by CIPKs in vitro. In addition, we report that phosphorylation of CBL1 by CIPK23 is also required for the CBL1-dependent enhancement of CIPK23 activity toward its substrate. Together, these data identify a novel general regulatory mechanism of CBL-CIPK complexes in that CBL phosphorylation at their flexible C terminus likely provokes conformational changes that enhance specificity and activity of CBL-CIPK complexes toward their target proteins.

A holistic phylogeny of the coronin gene family reveals an ancient origin of the tandem-coronin, defines a new subfamily, and predicts protein function.

BACKGROUND: Coronins belong to the superfamily of the eukaryotic-specific WD40-repeat proteins and play a role in several actin-dependent processes like cytokinesis, cell motility, phagocytosis, and vesicular trafficking. Two major types of coronins are known: First, the short coronins consisting of an N-terminal coronin domain, a unique region and a short coiled-coil region, and secondly the tandem coronins comprising two coronin domains. RESULTS: 723 coronin proteins from 358 species have been identified by analyzing the whole-genome assemblies of all available sequenced eukaryotes (March 2011). The organisms analyzed represent most eukaryotic kingdoms but also cover every taxon several times to provide a better statistical sampling. The phylogenetic tree of the coronin domains based on the Bayesian method is in accordance with the most recent grouping of the major kingdoms of the eukaryotes and also with the grouping of more recently separated branches. Based on this "holistic" approach the coronins group into four classes: class-1 (Type I) and class-2 (Type II) are metazoan/choanoflagellate specific classes, class-3 contains the tandem-coronins (Type III), and the new class-4 represents the coronins fused to villin (Type IV). Short coronins from non-metazoans are equally related to class-1 and class-2 coronins and thus remain unclassified. CONCLUSIONS: The coronin class distribution suggests that the last common eukaryotic ancestor possessed a single and a tandem-coronin, and most probably a class-4 coronin of which homologs have been identified in Excavata and Opisthokonts although most of these species subsequently lost the class-4 homolog. The most ancient short coronin already contained the trimerization motif in the coiled-coil domain.

Calcium-dependent modulation and plasma membrane targeting of the AKT2 potassium channel by the CBL4/CIPK6 calcium sensor/protein kinase complex.

Potassium (K(+)) channel function is fundamental to many physiological processes. However, components and mechanisms regulating the activity of plant K(+) channels remain poorly understood. Here, we show that the calcium (Ca(2+)) sensor CBL4 together with the interacting protein kinase CIPK6 modulates the activity and plasma membrane (PM) targeting of the K(+) channel AKT2 from Arabidopsis thaliana by mediating translocation of AKT2 to the PM in plant cells and enhancing AKT2 activity in oocytes. Accordingly, akt2, cbl4 and cipk6 mutants share similar developmental and delayed flowering phenotypes. Moreover, the isolated regulatory C-terminal domain of CIPK6 is sufficient for mediating CBL4- and Ca(2+)-dependent channel translocation from the endoplasmic reticulum membrane to the PM by a novel targeting pathway that is dependent on dual lipid modifications of CBL4 by myristoylation and palmitoylation. Thus, we describe a critical mechanism of ion-channel regulation where a Ca(2+) sensor modulates K(+) channel activity by promoting a kinase interaction-dependent but phosphorylation-independent translocation of the channel to the PM.

Switching off calcium-dependent inactivation in L-type calcium channels by an autoinhibitory domain.

The retinal L-type Ca2+ channel Cav1.4 is distinguished from all other members of the high voltage-activated (HVA) Ca2+ channel family by lacking Ca2+-calmodulin-dependent inactivation. In synaptic terminals of photoreceptors and bipolar cells, this feature is essential to translate graded membrane depolarizations into sustained Ca2+ influx and tonic glutamate release. The sequences conferring Ca2+-dependent inactivation (CDI) are conserved throughout the HVA calcium channel family, raising the question of how Cav1.4 manages to switch off CDI. Here, we identify an autoinhibitory domain in the distal C terminus of Cav1.4 that serves to abolish CDI. We show that this domain (ICDI, inhibitor of CDI) uncouples the molecular machinery conferring CDI from the inactivation gate by binding to the EF hand motif in the proximal C terminus. Deletion of ICDI completely restores Ca2+-calmodulin-mediated CDI in Cav1.4. CDI can be switched off again in the truncated Cav1.4 channel by coexpression of ICDI, indicating that ICDI works as an autonomous unit. Furthermore, we show that in the Cav1.2 l-type Ca2+-channel replacement of the distal C terminus by the corresponding sequence of Cav1.4 is sufficient to block CDI. This finding suggests that autoinhibition of CDI can be introduced principally into other Ca2+ channel types. Our data provide a previously undescribed perspective on the regulation of HVA calcium channels by Ca2+.

A novel mechanism of modulation of hyperpolarization-activated cyclic nucleotide-gated channels by Src kinase.

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-4) play a crucial role in the regulation of cell excitability. Importantly, they contribute to spontaneous rhythmic activity in brain and heart. HCN channels are principally activated by membrane hyperpolarization and binding of cAMP. Here, we identify tyrosine phosphorylation by Src kinase as another mechanism affecting channel gating. Inhibition of Src by specific blockers slowed down activation kinetics of native and heterologously expressed HCN channels. The same effect on HCN channel activation was observed in cells cotransfected with a dominant-negative Src mutant. Immunoprecipitation demonstrated that Src binds to and phosphorylates native and heterologously expressed HCN2. Src interacts via its SH3 domain with a sequence of HCN2 encompassing part of the C-linker and the cyclic nucleotide binding domain. We identified a highly conserved tyrosine residue in the C-linker of HCN channels (Tyr476 in HCN2) that confers modulation by Src. Replacement of this tyrosine by phenylalanine in HCN2 or HCN4 abolished sensitivity to Src inhibitors. Mass spectrometry confirmed that Tyr476 is phosphorylated by Src. Our results have functional implications for HCN channel gating. Furthermore, they indicate that tyrosine phosphorylation contributes in vivo to the fine tuning of HCN channel activity.

Patch testing with components of water-based metalworking fluids.

Water-based metalworking fluids (MWFs) may cause both irritant and allergic contact dermatitis. Several well-known MWF allergens are available for patch testing, but considering the wide variety of possible components used in MWF, our diagnostic arsenal covers only a small part of potential allergens. We therefore selected 13 frequently used MWF components that might be sensitizers and had not yet been tested routinely. In 5 centres, 233 dermatitis patients with present or past occupational exposure to MWF were patch tested with this and other panels. Only 7 patients showed positive reactions to the study panel. Allergic reactions to the emulsifier diglycolamine [syn. 2-(2-aminoethoxy) ethanol] were seen in 5 patients, and 1 patient each reacted positively to 2-amino-2-ethyl-1,3-propanediol (AEPD) and methyldiethanolamine (MDEA). Clinical relevance of the reactions to diglycolamine was unequivocally proven by its presence in the MWF from the patients' workplace in 3 cases. Diglycolamine seems to be an important MWF allergen, independently from monoethanolamine and diethanolamine. A test concentration of 1% petrolatum (pet.) appears to be appropriate. The importance of AEPD and MDEA as MWF allergens still remains to be established. The lack of positive test reactions to the other MWF components tested may be due to their low-sensitizing potential or too low a patch test concentration being used.