The disadvantages of being a hybrid during drought: A combined analysis of plant morphology, physiology and leaf proteome in maize.

A comparative analysis of various parameters that characterize plant morphology, growth, water status, photosynthesis, cell damage, and antioxidative and osmoprotective systems together with an iTRAQ analysis of the leaf proteome was performed in two inbred lines of maize (Zea mays L.) differing in drought susceptibility and their reciprocal F1 hybrids. The aim of this study was to dissect the parent-hybrid relationships to better understand the mechanisms of the heterotic effect and its potential association with the stress response. The results clearly showed that the four examined genotypes have completely different strategies for coping with limited water availability and that the inherent properties of the F1 hybrids, i.e. positive heterosis in morphological parameters (or, more generally, a larger plant body) becomes a distinct disadvantage when the water supply is limited. However, although a greater loss of photosynthetic efficiency was an inherent disadvantage, the precise causes and consequences of the original predisposition towards faster growth and biomass accumulation differed even between reciprocal hybrids. Both maternal and paternal parents could be imitated by their progeny in some aspects of the drought response (e.g., the absence of general protein down-regulation, changes in the levels of some carbon fixation or other photosynthetic proteins). Nevertheless, other features (e.g., dehydrin or light-harvesting protein contents, reduced chloroplast proteosynthesis) were quite unique to a particular hybrid. Our study also confirmed that the strategy for leaving stomata open even when the water supply is limited (coupled to a smaller body size and some other physiological properties), observed in one of our inbred lines, is associated with drought-resistance not only during mild drought (as we showed previously) but also during more severe drought conditions.

Arabidopsis proteome responses to the smoke-derived growth regulator karrikin.

Karrikins are butenolide plant growth regulators in smoke from burning plant material that have proven ability to promote germination and seedling photomorphogenesis. However, the molecular mechanisms underlying these processes are unclear. Here we provide the first proteome-wide analysis of early responses to karrikin in plants (Arabidopsis seedlings). Image analysis of two-dimensionally separated proteins, Rubisco-depleted proteomes and phosphoproteomes, together with LC-MS profiling, detected >1900 proteins, 113 of which responded to karrikin treatment. All the differentially abundant proteins (except HSP70-3) are novel karrikin-responders, and most are involved in photosynthesis, carbohydrate metabolism, redox homeostasis, transcription control, proteosynthesis, protein transport and processing, or protein degradation. Our data provide functionally complementary information to previous identifications of karrikin-responsive genes and evidence for a novel karrikin signalling pathway originating in chloroplasts. We present an updated model of karrikin signalling that integrates proteomic data and is supported by growth response observations. BIOLOGICAL SIGNIFICANCE: Karrikin has shown promising potential in agricultural applications, yet this process is poorly understood at the molecular level. To the best of our knowledge, this is the first survey of early global proteomic responses to karrikin in plants (Arabidopsis seedlings). The combination of label-free LC-MS profiling and 2-DE analyses provided highly sensitive snapshots of protein abundance and quantitative information on proteoform-level changes. These results present evidence of proteasome-independent karrikin signalling pathways and provide novel targets for detailed mechanistic studies using, e.g., mutants and transgenic plants.

The impact of heat stress targeting on the hormonal and transcriptomic response in Arabidopsis.

Targeting of the heat stress (HS, 40°C) to shoots, roots or whole plants substantially affects Arabidopsis physiological responses. Effective stress targeting was proved by determination of the expression of HS markers, HsfA2 and HSA32, which were quickly stimulated in the targeted organ(s), but remained low in non-stressed tissues for at least 2h. When shoots or whole plants were subjected to HS, a transient decrease in abscisic acid, accompanied by a small increase in active cytokinin levels, was observed in leaves, consistent with stimulation of transpiration, the main cooling mechanism in leaves. HS application targeted to part of plant resulted in a rapid stimulation of expression of components of cytokinin signaling pathway (especially of receptor genes) in the non-exposed tissues, which indicated fast inter-organ communication. Under all HS treatments, shoot apices responded by transient elevation of active cytokinin contents and stimulation of transcription of genes involved in photosynthesis and carbohydrate metabolism. Duration of this stimulation was negatively correlated with stress strength. The impact of targeted HS on the expression of 63 selected genes, including those coding regulatory 14-3-3 proteins, was compared. Stimulation of GRF9 (GRF14µ) in stressed organs after 2-6h may be associated with plant stress adaptation.

Characterization of a new human plasma cell leukemia cell line UHKT-944.

OBJECTIVE: A new interleukin-6 (IL-6)-dependent plasma cell leukemia cell line UHKT-944 was established from bone marrow cells derived from a 55-yr-old man with plasma cell leukemia. RESULTS: The cell line possesses phenotypic characteristics of plasma cells including the production of a monoclonal immunoglobulin IgA1-kappa. VH3-9 region of IgVH genes was rearranged and somatically hypermutated. The UHKT-944 cells were found to be negative for most of tested B-cell, T-cell, and myeloid markers. According to cytogenetic analysis, the cells were classified as near tetraploid with several numerical and structural abnormalities including the t(14;20) involving IgH locus. CONCLUSION: The established permanent plasma cell leukemia cell line is a suitable model for the study of cellular and molecular mechanisms of pathogenesis of this rare malignant disease.

Cytokinin modulates proteomic, transcriptomic and growth responses to temperature shocks in Arabidopsis.

As sessile organisms, plants must sense environmental conditions and adjust their growth and development processes accordingly, through adaptive responses regulated by various internal factors, including hormones. A key environmental factor is temperature, but temperature-sensing mechanisms are not fully understood despite intense research. We investigated proteomic responses to temperature shocks (15 min cold or heat treatments) with and without exogenous applications of cytokinin in Arabidopsis. Image and mass spectrometric analysis of the two-dimensionally separated proteins detected 139 differentially regulated spots, in which 148 proteins were identified, most of which have not been previously linked to temperature perception. More than 70% of the temperature-shock response proteins were modulated by cytokinin, mostly in a similar manner as heat shock. Data mining of previous transcriptomic datasets supported extensive interactions between temperature and cytokinin signalling. The biological significance of this finding was tested by assaying an independent growth response of Arabidopsis seedlings to heat stress: hypocotyl elongation. This response was strongly inhibited in mutants with deficiencies in cytokinin signalling or endogenous cytokinin levels. Thus, cytokinins may directly participate in heat signalling in plants. Finally, large proportions of both temperature-shock and cytokinin responsive proteomes co-localize to the chloroplast, which might therefore host a substantial proportion of the temperature response machinery.

Proteome and metabolome profiling of cytokinin action in Arabidopsis identifying both distinct and similar responses to cytokinin down- and up-regulation.

In plants, numerous developmental processes are controlled by cytokinin (CK) levels and their ratios to levels of other hormones. While molecular mechanisms underlying the regulatory roles of CKs have been intensely researched, proteomic and metabolomic responses to CK deficiency are unknown. Transgenic Arabidopsis seedlings carrying inducible barley cytokinin oxidase/dehydrogenase (CaMV35S>GR>HvCKX2) and agrobacterial isopentenyl transferase (CaMV35S>GR>ipt) constructs were profiled to elucidate proteome- and metabolome-wide responses to down- and up-regulation of CK levels, respectively. Proteome profiling identified >1100 proteins, 155 of which responded to HvCKX2 and/or ipt activation, mostly involved in growth, development, and/or hormone and light signalling. The metabolome profiling covered 79 metabolites, 33 of which responded to HvCKX2 and/or ipt activation, mostly amino acids, carbohydrates, and organic acids. Comparison of the data sets obtained from activated CaMV35S>GR>HvCKX2 and CaMV35S>GR>ipt plants revealed unexpectedly extensive overlaps. Integration of the proteomic and metabolomic data sets revealed: (i) novel components of molecular circuits involved in CK action (e.g. ribosomal proteins); (ii) previously unrecognized links to redox regulation and stress hormone signalling networks; and (iii) CK content markers. The striking overlaps in profiles observed in CK-deficient and CK-overproducing seedlings might explain surprising previously reported similarities between plants with down- and up-regulated CK levels.

Iron-induced changes in the proteome of Trichomonas vaginalis hydrogenosomes.

Iron plays a crucial role in metabolism as a key component of catalytic and redox cofactors, such as heme or iron-sulfur clusters in enzymes and electron-transporting or regulatory proteins. Limitation of iron availability by the host is also one of the mechanisms involved in immunity. Pathogens must regulate their protein expression according to the iron concentration in their environment and optimize their metabolic pathways in cases of limitation through the availability of respective cofactors. Trichomonas vaginalis, a sexually transmitted pathogen of humans, requires high iron levels for optimal growth. It is an anaerobe that possesses hydrogenosomes, mitochondrion-related organelles that harbor pathways of energy metabolism and iron-sulfur cluster assembly. We analyzed the proteomes of hydrogenosomes obtained from cells cultivated under iron-rich and iron-deficient conditions employing two-dimensional peptide separation combining IEF and nano-HPLC with quantitative MALDI-MS/MS. We identified 179 proteins, of which 58 were differentially expressed. Iron deficiency led to the upregulation of proteins involved in iron-sulfur cluster assembly and the downregulation of enzymes involved in carbohydrate metabolism. Interestingly, iron affected the expression of only some of multiple protein paralogues, whereas the expression of others was iron independent. This finding indicates a stringent regulation of differentially expressed multiple gene copies in response to changes in the availability of exogenous iron.

Novel human multiple myeloma cell line UHKT-893.

UNLABELLED: We established and characterized a new IL-6 dependent multiple myeloma (MM) cell line UHKT-893 from the bone marrow of a relapsed 57-year-old woman. RESULTS: Using nephelometry, cells with plasma cell phenotype and morphology were found to secrete IgG and free kappa (κ)-light chain of immunoglobulin. κ-Light chain was also recognized intracellularly by flow cytometry and by mass spectrometry. VH4-39 region of IgVH genes was rearranged and somatically hypermutated. Cytogenetic analysis of cells revealed new chromosome abnormalities in all breakpoints unique in both MM patients and cell lines - t(1;6), t(1;11), t(5;15), t(5;21), +der(11;15) and der(16). IL-6 independent subline UHKT-893a was established by adaptation to descending IL-6 concentration, while the original cell line keeps on maintaining its IL-6 dependency. CONCLUSION: The cell line provides a suitable material for cellular and molecular studies of tumor abnormalities, with potentially unique mutagenic features of myeloma disease. It may be utilized for human hybridoma construction and vaccine development. Both IL-6 dependent and independent cell clones represent an important model for studies of myeloma cell growth and resistance emerging during targeted therapy.

The physiology and proteomics of drought tolerance in maize: early stomatal closure as a cause of lower tolerance to short-term dehydration?

Understanding the response of a crop to drought is the first step in the breeding of tolerant genotypes. In our study, two maize (Zea mays L.) genotypes with contrasting sensitivity to dehydration were subjected to moderate drought conditions. The subsequent analysis of their physiological parameters revealed a decreased stomatal conductance accompanied by a slighter decrease in the relative water content in the sensitive genotype. In contrast, the tolerant genotype maintained open stomata and active photosynthesis, even under dehydration conditions. Drought-induced changes in the leaf proteome were analyzed by two independent approaches, 2D gel electrophoresis and iTRAQ analysis, which provided compatible but only partially overlapping results. Drought caused the up-regulation of protective and stress-related proteins (mainly chaperones and dehydrins) in both genotypes. The differences in the levels of various detoxification proteins corresponded well with the observed changes in the activities of antioxidant enzymes. The number and levels of up-regulated protective proteins were generally lower in the sensitive genotype, implying a reduced level of proteosynthesis, which was also indicated by specific changes in the components of the translation machinery. Based on these results, we propose that the hypersensitive early stomatal closure in the sensitive genotype leads to the inhibition of photosynthesis and, subsequently, to a less efficient synthesis of the protective/detoxification proteins that are associated with drought tolerance.

Electrophoretic mobility of cardiac myosin heavy chain isoforms revisited: application of MALDI TOF/TOF analysis.

The expression of two cardiac myosin heavy chain (MyHC) isoforms in response to the thyroid status was studied in left ventricles (LVs) of Lewis rats. Major MyHC isoform in euthyroid and hyperthyroid LVs had a higher mobility on SDS-PAGE, whereas hypothyroid LVs predominantly contained a MyHC isoform with a lower mobility corresponding to that of the control soleus muscle. By comparing the MyHC profiles obtained under altered thyroid states together with the control soleus, we concluded that MyHCα was represented by the lower band with higher mobility and MyHCß by the upper band. The identity of these two bands in SDS-PAGE gels was confirmed by western blot and mass spectrometry. Thus, in contrast to the literature data, we found that the MyHCα possessed a higher mobility rate than the MyHCß isoform. Our data highlighted the importance of the careful identification of the MyHCα and MyHCß isoforms analyzed by the SDS-PAGE.

The core components of organelle biogenesis and membrane transport in the hydrogenosomes of Trichomonas vaginalis.

Trichomonas vaginalis is a parasitic protist of the Excavata group. It contains an anaerobic form of mitochondria called hydrogenosomes, which produce hydrogen and ATP; the majority of mitochondrial pathways and the organellar genome were lost during the mitochondrion-to-hydrogenosome transition. Consequently, all hydrogenosomal proteins are encoded in the nucleus and imported into the organelles. However, little is known about the membrane machineries required for biogenesis of the organelle and metabolite exchange. Using a combination of mass spectrometry, immunofluorescence microscopy, in vitro import assays and reverse genetics, we characterized the membrane proteins of the hydrogenosome. We identified components of the outer membrane (TOM) and inner membrane (TIM) protein translocases include multiple paralogs of the core Tom40-type porins and Tim17/22/23 channel proteins, respectively, and uniquely modified small Tim chaperones. The inner membrane proteins TvTim17/22/23-1 and Pam18 were shown to possess conserved information for targeting to mitochondrial inner membranes, but too divergent in sequence to support the growth of yeast strains lacking Tim17, Tim22, Tim23 or Pam18. Full complementation was seen only when the J-domain of hydrogenosomal Pam18 was fused with N-terminal region and transmembrane segment of the yeast homolog. Candidates for metabolite exchange across the outer membrane were identified including multiple isoforms of the ß-barrel proteins, Hmp35 and Hmp36; inner membrane MCF-type metabolite carriers were limited to five homologs of the ATP/ADP carrier, Hmp31. Lastly, hydrogenosomes possess a pathway for the assembly of C-tail-anchored proteins into their outer membrane with several new tail-anchored proteins being identified. These results show that hydrogenosomes and mitochondria share common core membrane components required for protein import and metabolite exchange; however, they also reveal remarkable differences that reflect the functional adaptation of hydrogenosomes to anaerobic conditions and the peculiar evolutionary history of the Excavata group.

Searching for major urinary proteins (MUPs) as chemosignals in urine of subterranean rodents.

Chemosensory information mediates behavior in many rodent genera. Major Urinary Proteins (MUPs) facilitate chemical communication in some species of mice. We sought to demonstrate the importance of MUPs in chemosignaling across a range of rodent genera that live in different habitats and social structures. We analyzed urine from three subterranean rodent genera from different continents, and with diverse social systems: eusocial Zambian mole-rats (Fukomys), solitary Israeli blind mole rats (Spalax), and social Chilean coruros (Spalacopus). 2D gel electrophoresis revealed low levels of protein, with sequences similar to aphrodisin, in Fukomys mole-rat urine, but no MUPs in urine of any of the studied species. Previous research demonstrated that subjects from the tested genera responded differentially to odors indicating transmission of individuality, family/colony or population, species, and reproductive state in secretions and excretions of conspecifics. This extends, to subterranean rodents, the evidence that rodent species can successfully transmit and receive chemosignals without the necessity of MUPs.

The minimal proteome in the reduced mitochondrion of the parasitic protist Giardia intestinalis.

The mitosomes of Giardia intestinalis are thought to be mitochondria highly-reduced in response to the oxygen-poor niche. We performed a quantitative proteomic assessment of Giardia mitosomes to increase understanding of the function and evolutionary origin of these enigmatic organelles. Mitosome-enriched fractions were obtained from cell homogenate using Optiprep gradient centrifugation. To distinguish mitosomal proteins from contamination, we used a quantitative shot-gun strategy based on isobaric tagging of peptides with iTRAQ and tandem mass spectrometry. Altogether, 638 proteins were identified in mitosome-enriched fractions. Of these, 139 proteins had iTRAQ ratio similar to that of the six known mitosomal markers. Proteins were selected for expression in Giardia to verify their cellular localizations and the mitosomal localization of 20 proteins was confirmed. These proteins include nine components of the FeS cluster assembly machinery, a novel diflavo-protein with NADPH reductase activity, a novel VAMP-associated protein, and a key component of the outer membrane protein translocase. None of the novel mitosomal proteins was predicted by previous genome analyses. The small proteome of the Giardia mitosome reflects the reduction in mitochondrial metabolism, which is limited to the FeS cluster assembly pathway, and a simplicity in the protein import pathway required for organelle biogenesis.

Three metallothionein isoforms and sequestration of intracellular silver in the hyperaccumulator Amanita strobiliformis.

Metallothioneins (MTs) are cysteine-rich peptides involved in heavy metal tolerance of many eukaryotes. Here, we examined their involvement in intracellular binding of silver (Ag) in the ectomycorrhizal fungus Amanita strobiliformis. The Ag complexes and their peptide ligands were characterized using chromatography and mass spectrometry. The full-length coding sequences obtained from a cDNA library were used for complementation assays in yeast mutant strains. Abundance of respective transcripts in A. strobiliformis was measured by quantitative real-time reverse-transcribed polymerase chain reaction (qRT-PCR). Ag-speciation analyses showed that intracellular Ag was in wild-grown fruit bodies and cultured extraradical mycelia of A. strobiliformis sequestered by metallothioneins. The determined sequence of the peptide facilitated isolation of three cDNA clones, AsMT1a, AsMT1b and AsMT1c. These encode isomorphic MTs consisting of 34 amino acid residues and sharing 82% identity. In mycelia the expression of AsMT1s is induced by Ag. All AsMT1s expressed in yeasts complemented hypersensitivity of mutants to cadmium (Cd) and copper (Cu) and formed Ag complexes. Only the Ag-AsMT1a complex was detected in the A. strobiliformis fruit body in which AsMT1a was the prevailing transcript. The present study identified the existence of metallothionein isoforms in ectomycorrhizal fungi. We demonstrated that intracellular sequestration of Ag in fruit bodies and mycelia of hyperaccumulating A. strobiliformis is dominated by metallothioneins.

Cytoskeleton-associated large RNP complexes in tobacco male gametophyte (EPPs) are associated with ribosomes and are involved in protein synthesis, processing, and localization.

The progamic phase of male gametophyte development involves activation of synthetic and catabolic processes required for the rapid growth of the pollen tube. It is well-established that both transcription and translation play an important role in global and specific gene expression patterns during pollen maturation. On the contrary, germination of many pollen species has been shown to be largely independent of transcription but vitally dependent on translation of stored mRNAs. Here, we report the first structural and proteomic data about large ribonucleoprotein particles (EPPs) in tobacco male gametophyte. These complexes are formed in immature pollen where they contain translationally silent mRNAs. Although massively activated at the early progamic phase, they also serve as a long-term storage of mRNA transported along with the translational machinery to the tip region. Moreover, EPPs were shown to contain ribosomal subunits, rRNAs and a set of mRNAs. Presented results extend our view of EPP complexes from mere RNA storage and transport compartment in particular stages of pollen development to the complex and well-organized machinery devoted to mRNA storage, transport and subsequent controlled activation resulting in protein synthesis, processing and precise localization. Such an organization is extremely useful in fast tip-growing pollen tube. There, massive and orchestrated protein synthesis, processing, and transport must take place in accurately localized regions. Moreover, presented complex role of EPPs in tobacco cytoplasmic mRNA and protein metabolism makes them likely to be active in another plant species too. Expression of vast majority of the closest orthologues of EPP proteins also in Arabidopsis male gametophyte further extends this concept from tobacco to Arabidopsis, the model species with advanced tricellular pollen.