Proteomic characterization of Phragmites communis in ecotypes of swamp and desert dune

Phragmites communis Trin. (common reed) is a recognized model plant for studying its adaptation to contrasting and harsh environments. To understand the inherent molecular basis for its remarkable resistance to combined stresses, we performed a comprehensive proteomic analysis of the leaf proteins from two ecotypes, i.e. swamp and desert dune, naturally growing in the desert region of northwestern China. First, a proteome reference map of Phragmites was established based on the swamp ecotype. Proteins were resolved by 2‐D/SDS‐PAGE and identified by MALDI‐TOF/TOF MS. In total, 177 spots were identified corresponding to 51 proteins. The major proteins identified are proteins involved in photosynthesis, glutathione and ascorbic acid metabolism as well as protein synthesis and quality control. Second, the 2‐DE profiles of the two ecotypes were compared quantitatively via DIGE analysis. Compared with swamp ecotype, 51 proteins spots are higher‐expressed and 58 protein spots are lower‐expressed by twofold or more in desert dune ecotype. Major differences were found for the proteins involved in light reaction of photosynthesis, protein biosynthesis and quality control and antioxidative reactions. The physiological significance of such differences is discussed in the context of a flow of complex events in relation to plant adaptation to combined environmental stresses.     

Proteome analysis differentiates between two highly homologues germin-like proteins in Arabidopsis thaliana ecotypes Col-0 and Ws-2

A proteome approach based on 2-D gel electrophoresis (2-DE) was used to compare the protein patterns of the Arabidopsis ecotypes Col-0 and Ws-2. In leaf extracts a pair of protein spots were found to be diagnostic for each of the lines. Both pairs of spots were identified as closely related germin-like proteins differing in only one amino acid by using peptide mass finger printing of tryptic digests and by gaining additional data from post-source decay spectra in the MALDI-TOF analysis. Western blot analysis after separation of protein extracts by 2-DE confirmed results from Coomassie blue-stained gels and revealed additional immunoreactive spots for both ecotypes most likely representing dimers of the spots first identified. Western blot analysis and mass spectrometrical identification of the corresponding weakly stained protein in Coomassie blue-stained gels of the ecotype Col-0 also demonstrated for the first time the occurrence of AtGER3 protein in root extracts. Our results demonstrate the capacity of proteome analysis to analyse and distinguish closely related members of large protein families.     

Proteomic analysis of F1 hybrids and intermediate variants in a Littorina saxatilis hybrid zone

Proteomic analysis was carried out on the Crab (upper-shore) and Wave (lower-shore) ecotypes of Littorina saxatilis from a hybrid zone at Silleiro Cape, Spain. Proteome profiles of individual snails were obtained. Protein expression in F₁ hybrid snails bred in the laboratory and snails with intermediate shell phenotypes collected from the mid-shore were compared with Crab and Wave ecotypes using analytical approaches used to study dominance. Multivariate analysis over many protein spots showed that the F₁ snails are distinct from both ecotypes but closer to the Wave ecotype. The intermediate snails are highly variable, some closer to the Crab and others to the Wave ecotype. Considered on a protein by protein basis, some proteins are significantly closer in expression to the Crab and others to the Wave ecotype for both F₁ and intermediate snails. Furthermore, a significant majority of proteins were closer in expression to the Wave ecotype for the F₁, consistent with the multivariate analysis. No such significant majority toward either the Crab or Wave ecotype was observed for the intermediate snails. The closer similarity of F₁ and Wave ecotype expression patterns could be the result of similar selective pressures in the similar mid-shore and low-shore environments. For a significantly larger number of proteins, intermediate snails were closer in expression to the ecotype having the lower expression, for both Crab and Wave ecotypes. This is somewhat unexpected as lower expression might be expected to be an indication of impairment of function and lower fitness. Proteomic analysis could be important for the identification of candidate proteins useful for gaining improved understanding of adaptation and barriers to gene flow in hybrid zones.     

Recent stable insertion of mitochondrial DNA into an Arabidopsis polyubiquitin gene by nonhomologous recombination.

Sequence analysis of a newly identified polyubiquitin gene (UBQ13) from the Columbia ecotype of Arabidopsis thaliana revealed that the gene contained a 3.9-kb insertion in the coding region. All subclones of the 3.9-kb insert hybridized to isolated mitochondrial DNA. The insert was found to consist of at least two, possibly three, distinct DNA segments from the mitochondrial genome. A 590-bp region of the insert is nearly identical to the Arabidopsis mitochondrial nad1 gene. UBQ13 restriction fragments in total cellular DNA from ecotypes Ler, No-0, Be-0, WS, and RLD were identified and, with the exception of Be-0, their sizes were equivalent to that predicted from the corresponding ecotype Columbia UBQ13 restriction fragment without the mitochondrial insert. Isolation by polymerase chain reaction and sequence determination of UBQ13 sequences from the other ecotypes showed that all lacked the mitochondrial insert. All ecotypes examined, except Columbia, contain intact open reading frames in the region of the insert, including four ubiquitin codons which Columbia lacks. This indicates that the mitochondrial DNA in UBQ13 in ecotype Columbia is the result of an integration event that occurred after speciation of Arabidopsis rather than a deletion event that occurred in all ecotypes except Columbia. This stable movement of mitochondrial DNA to the nucleus is so recent that there are few nucleotide changes subsequent to the transfer event. This allows for precise analysis of the sequences involved and elucidation of the possible mechanism. The presence of intron sequences in the transferred nucleic acid indicates that DNA was the transfer intermediate. The lack of sequence identity between the integrating sequence and the target site, represented by the other Arabidopsis ecotypes, suggests that integration occurred via nonhomologus recombination. This nuclear/organellar gene transfer event is strikingly similar to the experimentally accessible process of nuclear integration of introduced heterologous DNA.     

Heat induced changes in protein expression profiles of Norway spruce (Picea abies) ecotypes from different elevations

Although tree species typically exhibit low genetic differentiation between populations, ecotypes adapted to different environmental conditions can vary in their capacity to withstand and recover from environmental stresses like heat stress. Two month old seedlings of a Picea abies ecotype adapted to high elevation showed lower level of thermotolerance and higher level of tolerance to oxidative stress relative to a low elevation ecotype. Protein expression patterns following exposure to severe heat stress of the two ecotypes were compared by means of 2-DE. Several proteins exhibiting ecotype and tissue specific expression were identified by MS/MS. Among them, small heat shock proteins of the HSP 20 family and proteins involved in protection from oxidative stress displayed qualitative and quantitative differences in expression between the ecotypes correlated with the observed phenotypic differences. On the basis of these results, it can be speculated that the observed interpopulation polymorphism of protein regulation in response to heat stress could underlie their different capacities to withstand and recover from heat stress. These local adaptations are potentially relevant for the species adaptation to the conditions predicted by the current models for climate change.     

Proteomic evidence of a paedomorphic evolutionary process within a marine snail species: a strategy for adapting to extreme ecological conditions?

The exposed and sheltered ecotypes of the marine snail Littorina saxatilis from European rocky shores are considered a key model system to study adaptation and ecological speciation. Previous studies showed that two ecotypes (RB and SU) of this species in NW Spain have adapted differently to different shore levels and microhabitats. In order to understand how this divergent adaptive process has been accomplished, we followed a quantitative proteomic approach to investigate the proteome variation in a number of different biological factors, that is, ecotype, ontogeny and their interactions. This approach allowed testing the hypothesis that one of the ecotypes has evolved by paedomorphosis, and also whether or not the molecular mechanisms related to ecotype differentiation are set up in early developmental stages. Additionally, the identification of some candidate proteins using mass spectrometry provides some functional insights into these evolutionary processes. Results from this study provided evidence of higher ontogenetic differentiation at proteome level in the RB (metamorphic) than in SU (paedomorphic) ecotype that point to the possibility of juvenile stage retention in this latter ecotype. The level of protein expression (proteome) differences between ecotypes maintained nearly constant from late embryonic stages to adulthood, although some proteins involved in these changes considerably differed in embryonic compared to other ontogenetic stages. Paedomorphosis may be the evolutionary response of the SU ecotype of solving the trade‐off during sexually immaturity that is caused by the evolution of small size arising from adaptation to the wave‐exposed habitat. Some potential candidate genes of adaptation related to energetic metabolism have been identified, providing a promising baseline for future functional analyses.     

Aconitate hydratase of mammals under oxidative stress

Data on the structure, functions, regulation of activity, and expression of cytosolic and mitochondrial aconitate hydratase isoenzymes of mammals are reviewed. The role of aconitate hydratase and structurally similar iron-regulatory protein in maintenance of homeostasis of cell iron is described. Information on modifications of the aconitate hydratase molecule and changes in expression under oxidative stress is generalized. The role of aconitate hydratase in the pathogenesis of some diseases is considered.     

<Emphasis Type="Italic">Arabidopsis TTR1</Emphasis> causes LRR-dependent lethal systemic necrosis,rather than systemic acquired resistance,to Tobacco ringspot virus

Most Arabidopsis ecotypes display tolerance to the Tobacco ringspot virus (TRSV), but a subset of Arabidopsis ecotypes, including Estland (Est), develop lethal systemic necrosis (LSN), which differs from the localized hypersensitive responses (HRs) or systemic acquired resistance (SAR) characteristic of incompatible reactions. Neither viral replication nor the systemic movement of TRSV was restricted in tolerant or sensitive Arabidopsis ecotypes; therefore, the LSN phenotype shown in the sensitive ecotypes might not be due to viral accumulation. In the present study, we identified the Est TTR1 gene (tolerance to Tobacco ringspot virus 1) encoding a TIR-NBS-LRR protein that controls the ecotype-dependent tolerant/sensitive phenotypes by a map-based cloning method. The tolerant Col-0 ecotype Arabidopsis transformed with the sensitive Est TTR1 allele developed an LSN phenotype upon TRSV infection, suggesting that the Est TTR1 allele is dominant over the tolerant ttr1 allele of Col-0. Multiple sequence alignments of 10 tolerant ecotypes from those of eight sensitive ecotypes showed that 10 LRR amino acid polymorphisms were consistently distributed across the TTR1/ttr1 alleles. Site-directed mutagenesis of these amino acids in the LRR region revealed that two sites, L956S and K1124Q, completely abolished the LSN phenotype. VIGS study revealed that TTR1 is dependent on SGT1, rather than EDS1. The LSN phenotype by TTR1 was shown to be transferred to Nicotiana benthamiana, demonstrating functional conservation of TTR1 across plant families, which are involved in SGT-dependent defense responses, rather than EDS1-dependent signaling pathways.     

Proteomic comparison between two marine snail ecotypes reveals details about the biochemistry of adaptation

The proteomic changes occurring during speciation are fundamental to understand this process, though they have been rarely addressed until present. Therefore, we compared the proteome of two ecotypes (RB and SU) of the marine snail Littorina saxatilis, a case of sympatric incomplete speciation, originated as a byproduct of adaptation to distinct habitats. Thus, the RB ecotype is able to resist stresses of desiccation and temperature on the upper shore, whereas the SU ecotype defies strong physical disturbances due to wave action. Qualitative analyses of 2-DE gels demonstrated 21 proteins differentially expressed (1.4% of the proteome, 1.2% after considering type-I errors), while quantitative changes accounted for differences in 22 spots (16% of the proteome, 11% after considering type-I errors). These results suggest that adaptative phenotypic plasticity, natural selection, or both maintain these ecotypes in sympatry. Among the proteins identified by MS, we found that fructose-bisphosphate aldolase and arginine kinase were up-regulated in the SU ecotype, suggesting an enhancement of the level of energy available as ATP, in order to withstand its wave-exposed habitat.     

P uptake characteristics and root morphological responses in the mining ecotype of Polygonum hydropiper under high organic P media

Understanding plant phosphorus (P) assimilation and its root morphological responses is important to acquire an ideal material for remediation of P-enriched environments. Pot experiments were conducted to explore P accumulation and root morphological traits in a mining ecotype (ME) and non-mining ecotype (NME) of Polygonum hydropiper under different organic P (Po) sources (G1P, AMP, ATP, IHP) and inorganic P (Pi) source (KH₂PO₄), and also their responses to a high level of IHP for different growth periods. Both ecotypes showed higher biomass in Pi and IHP treatments than other Po sources. P accumulation in seedlings were in the order of Pi > IHP > other Po media. Extending the growth period increased biomass and P accumulation in both ecotypes. The ME demonstrated 1.11–1.46 times higher P accumulation than the NME. Seedlings fed with IHP demonstrated significantly greater morphological parameters of fine, medium, and thick roots compared to other Po sources. Total root length, surface area, and volume of both ecotypes significantly increased with the prolonged growth period. The ME has a higher ability to develop root system and exhibits better distribution of fine roots to enhance P accumulation from high P media, and thus it is a worthy material for P-phytoextraction.     

Proteomic analysis of the anterior cingulate cortex in the major psychiatric disorders: Evidence for disease-associated changes

Abnormalities of the anterior cingulate cortex have previously been described in schizophrenia, major depressive disorder and bipolar disorder. In this study 2-DE was performed followed by mass spectrometric sequencing to identify disease-specific protein changes within the anterior cingulate cortex in these psychiatric disorders. The 2-DE system comprised IPGs 4-7 and 6-9 in the first, IEF dimension and SDS-PAGE in the second dimension. Resultant protein spots were compared between control and disease groups. Statistical analysis indicated that 35 spots were differentially expressed in one or more groups. Proteins comprising 26 of these spots were identified by mass spectroscopy. These represented 19 distinct proteins; aconitate hydratase, malate dehydrogenase, fructose bisphosphate aldolase A, ATP synthase, succinyl CoA ketoacid transferase, carbonic anhydrase, alpha- and beta-tubulin, dihydropyrimidinase-related protein-1 and -2, neuronal protein 25, trypsin precursor, glutamate dehydrogenase, glutamine synthetase, sorcin, vacuolar ATPase, creatine kinase, albumin and guanine nucleotide binding protein beta subunit. All but three of these proteins have previously been associated with the major psychiatric disorders. These findings provide support for the view that cytoskeletal and mitochondrial dysfunction are important components of the neuropathology of the major psychiatric disorders.     

Natural Variation in Stomatal Responses to Environmental Changes among Arabidopsis thaliana Ecotypes

Stomata are small pores surrounded by guard cells that regulate gas exchange between plants and the atmosphere. Guard cells integrate multiple environmental signals and control the aperture width to ensure appropriate stomatal function for plant survival. Leaf temperature can be used as an indirect indicator of stomatal conductance to environmental signals. In this study, leaf thermal imaging of 374 Arabidopsis ecotypes was performed to assess their stomatal responses to changes in environmental CO2 concentrations. We identified three ecotypes, Köln (Kl-4), Gabelstein (Ga-0), and Chisdra (Chi-1), that have particularly low responsiveness to changes in CO2 concentrations. We next investigated stomatal responses to other environmental signals in these selected ecotypes, with Col-0 as the reference. The stomatal responses to light were also reduced in the three selected ecotypes when compared with Col-0. In contrast, their stomatal responses to changes in humidity were similar to those of Col-0. Of note, the responses to abscisic acid, a plant hormone involved in the adaptation of plants to reduced water availability, were not entirely consistent with the responses to humidity. This study demonstrates that the stomatal responses to CO2 and light share closely associated signaling mechanisms that are not generally correlated with humidity signaling pathways in these ecotypes. The results might reflect differences between ecotypes in intrinsic response mechanisms to environmental signals.