Genetic variation and drought response in two Populus × euramericana genotypes through 2-DE proteomic analysis of leaves from field and glasshouse cultivated plants

Genotype and water deficit effects on leaf 2-DE protein profiles of two Populus deltoides × Populus nigra, cv. ‘Agathe_F’ and ‘Cima’, were analysed over a short-term period of 18 days in glasshouse using 4-month-old rooted cuttings and over a long-lasting period of 86 days in open field using 4-year-old rooted cuttings. Leaf proteomes were analyzed using two-dimensional gel electrophoresis, and proteins were identified after database searching from MS peptide spectra.A reliable genotype effect was observed in the leaf proteome over experiment locations, water regimes and sampling dates. Quantitative differences between genotypes were found. Most of them corresponded to proteins matching isoforms or post-translational modification variants. However, ‘Cima’ displayed the highest abundance of antioxidant enzymes.In response to water deficit, about 10% of the reproducible spots significantly varied regardless of the experiment location, among which about 25% also displayed genotype-dependent variations. As a whole, while ‘Cima’ differed from ‘Agathe_F’ by increased abundance of enzymes involved in photorespiration and in oxidative stress, ‘Agathe_F’ was mainly differentiated by increased abundance of enzymes involved in photosynthesis.     

Comparative analysis of proteome changes induced by the two spotted spider mite Tetranychus urticae and methyl jasmonate in citrus leaves

Citrus plants are currently facing biotic and abiotic stresses. Therefore, the characterization of molecular traits involved in the response mechanisms to stress could facilitate selection of resistant varieties. Although large cDNA microarray profiling has been generated in citrus tissues, the available protein expression data are scarce. In this study, to identify differentially expressed proteins in Citrus clementina leaves after infestation by the two-spotted spider mite Tetranychus urticae, a proteome comparison was undertaken using two-dimensional gel electrophoresis. The citrus leaf proteome profile was also compared with that of leaves treated over 0-72 h with methyl jasmonate, a compound playing a key role in the defense mechanisms of plants to insect/arthropod attack. Significant variations were observed for 110 protein spots after spider mite infestation and 67 protein spots after MeJA treatments. Of these, 50 proteins were successfully identified by liquid chromatography-mass spectrometry-tandem mass spectrometry. The majority constituted photosynthesis- and metabolism-related proteins. Five were oxidative stress associated enzymes, including phospholipid glutathione peroxidase, a salt stressed associated protein, ascorbate peroxidase and Mn-superoxide dismutase. Seven were defense-related proteins, such as the pathogenesis-related acidic chitinase, the protease inhibitor miraculin-like protein, and a lectin-like protein. This is the first report of differentially regulated proteins after T. urticae attack and exogenous MeJA application in citrus leaves.     

Phytosiderophore release by wheat genotypes differing in zinc deficiency tolerance grown with Zn-free nutrient solution as affected by salinity

There is limited information concerning the effect of salinity on phytosiderophores exudation from wheat roots. The aim of this hydroponic experiment was to investigate the effect of salinity on phytosiderophore release by roots of three bread wheat genotypes differing in Zn efficiency (Triticum aestivum L. cvs. Rushan, Kavir, and Cross) under Zn deficiency conditions. Wheat seedlings were transferred to Zn-free nutrient solutions and exposed to three salinity levels (0, 60, and 120 mM NaCl). The results indicated that Cross and Rushan genotypes exuded more phytosiderophore than did the Kavir genotype. Our findings suggest that the adaptive capacity of Zn-efficient ‘Cross’ and ‘Rushan’ wheat genotypes to Zn deficiency is due partly to the higher amounts of phytosiderophore release. Only 15 days of Zn deficiency stress was sufficient to distinguish between Zn-efficient (Rushan and Cross) and Zn-inefficient (Kavir) genotypes, with the former genotypes exuding more phytosiderophore than the latter. Higher phytosiderophore exudation under Zn deficiency conditions was accompanied by greater Fe transport from root to shoot. The maximum amount of phytosiderophore was exuded at the third week in ‘Cross’ and at the fourth week in ‘Kavir’ and ‘Rushan’. For all three wheat genotypes, salinity stress resulted in higher amounts of phytosiderophore exuded by the roots. In general, for ‘Kavir’, the largest amount of phytosiderophore was exuded from the roots at the highest salinity level (120 mM NaCl), while for ‘Cross’ and ‘Rushan’, no significant difference was found in phytosiderophore exudation between the 60 and 120 mM NaCl treatments. More investigation is needed to fully understand the physiology of elevated phytosiderophore release by Zn-deficient wheat plants under salinity conditions.     

Rye oxidative stress under long term Al exposure

Aluminium (Al) toxicity decreases plant growth. Secale cereale L. is among the most Al-tolerant crop species. In order to study the response to Al-long term exposure, two rye genotypes with different Al sensitivity (‘D. Zlote’ and ‘Riodeva’) were exposed to 1.11 and 1.85 mM Al and the antioxidant responses were followed for 2 and 3 weeks in roots and leaves. Al toxicity signals, such as a severe decrease in root growth, occurred sooner in ‘Riodeva.’ The antioxidant response was dependent on the genotype, the organ, Al concentration and the exposure period. Al-exposed roots of ‘D. Zlote’ showed earlier enhancements of APX, SOD and G-POX activities than those of ‘Riodeva.’ ‘D. Zlote’ roots showed stimulation of the AsA–GSH cycle after the second week (when root growth inhibition was less severe), while later (when severe root growth inhibition was observed), oxidation of AsA and GSH pools was observed. In leaves of both genotypes, CAT, SOD and G-POX activities increased with Al exposure. In these leaves, the effect of AsA–GSH was time dependent, with maximum oxidation at the second week, followed by recovery. We confirmed that the oxidation state of AsA and GSH pools is involved in the detoxification of Al-induced oxidative stress. Moreover, our data demonstrate that the production of ROS does not correlate with the Al-induced root growth decrease. Finally, the differences observed over time indicate that long term exposure may provide additional information on rye sensitivity to Al, and contribute to a better understanding of this species’ mechanisms of Al tolerance.     

Tetraploid citrus rootstocks are more tolerant to salt stress than diploid

Citrus trees are subject to several abiotic constraints such as salinity. Providing new rootstocks more tolerant is thus a requirement. In this article, we investigated salt stress tolerance of three tetraploid rootstock genotypes when compared to their respective diploid rootstocks (Poncirus trifoliata, Carrizo citrange, Cleopatra mandarin). Plant growth, leaf fall and ion contents were investigated. At the end of the experiment, leaf fall was observed only for diploid Poncirus trifoliata plants as well as chlorosis symptoms for Poncirus trifoliata and Carrizo citrange diploid plants. The diploid Cleopatra mandarin plants growth rate was not affected by salt stress and has even been increased for tetraploid Cleopatra mandarin. Ion contents investigation has shown lower accumulations of chloride ions in leaves of the tetraploid plants when compared to diploid plants. Our results suggest that citrus tetraploid rootstocks are more tolerant to salt stress than their corresponding diploid. To cite this article: B. Saleh et al., C. R. Biologies 331 (2008).     

Assessment of agroeconomic indices in polycultures of lettuce, rocket and carrot through uni- and multivariate approaches in semi-arid Brazil

The polyculture among vegetables is an activity that to have good results, needs a proper planning. Although it often requires more labor, has several advantages over monoculture, among them is that polycultures are generally are more productive, provide with productivity of various plant constituents and a more balanced human diet, contribute to economic return, economic and yield stability, social benefits and farmer's direct participation in decision-making. The objective of this study was to evaluate agroeconomic indices of polycultures derived from the combination of two cultivars of lettuce with two cultivars of rocket in two cultures strip-intercropped with carrot cultivar ‘Brasilia’ through uni-multivariate approaches in semi-arid Brazil. The experimental design used was of randomized complete blocks with five replications and the treatments arranged in a factorial scheme of 2 × 2. The treatments consisted of the combination of two lettuce cultivars (Babá de Verão and Tainá) with two rocket cultivars (Cultivada and Folha Larga) in two cultures associated with carrot cv. Brasília. In each block were grown plots with two lettuce cultivars and two rocket cultivars, and carrot in sole crop. In each system was determined the lettuce leaf yield, rocket green mass yield and carrot commercial yield. Agrieconomic indices such as operational cost, gross and net income, monetary advantage, rate of return, profit margin, land equivalent ratio and yield efficiency for DEA were used to measure the efficiency of intercropping systems. In the bicropping of lettuce and rocket associated with carrot cv. ‘Brasilia’, suggests the use of lettuce cultivar ‘Tainá’ combined with rocket cultivars ‘Cultivada’ or ‘Folha Larga’. It was observed significant effect of lettuce cultivars in the evaluation of polycultures of lettuce, carrot and rocket, with strong expression for the lettuce cultivar ‘Tainá’. Both uni- and multivariate approaches were effective in the discrimination of the best polycultures.     

Comparative proteomic analysis of seedling leaves of different salt tolerant soybean genotypes

Salinity is one of the major environmental constraints limiting yield of crop plants in many semi-arid and arid regions around the world. To understand responses in soybean seedling to salt stress at proteomic level, the extracted proteins from seedling leaves of salt-sensitive genotype Jackson and salt-tolerant genotype Lee 68 under 150 mM NaCl stress for 1, 12, 72 and 144 h, respectively, were analyzed by 2-DE. Approximately 800 protein spots were detected on 2-DE gels. Among them, 91 were found to be differently expressed, with 78 being successfully identified by MALDI-TOF-TOF. The identified proteins were involved in 14 metabolic pathways and cellular processes. Based on most of the 78 salt-responsive proteins, a salt stress-responsive protein network was proposed. This network consisted of several functional components, including balancing between ROS production and scavenging, accelerated proteolysis and reduced biosynthesis of proteins, impaired photosynthesis, abundant energy supply and enhanced biosynthesis of ethylene. Salt-tolerant genotype Lee 68 possessed the ability of higher ROS scavenging, more abundant energy supply and ethylene production, and stronger photosynthesis than salt-sensitive genotype Jackson under salt stress, which may be the major reasons why it is more salt-tolerant than Jackson.     

Responses of antioxidant gene, protein and enzymes to salinity stress in two genotypes of perennial ryegrass (Lolium perenne) differing in salt tolerance

Salinity could damage cellular membranes through overproduction of reactive oxygen species (ROS), while antioxidant capacities play a vital role in protecting plants from salinity caused oxidative damages. The objective of this study was to investigate the toxic effect of salt on the antioxidant enzyme activities, isoforms and gene expressions in perennial ryegrass (Lolium perenne L.). Salt-tolerant ‘Quickstart II’ and salt-sensitive ‘DP1′ were subjected to 0 and 250 mM NaCl for 12 d. Salt stress increased the content of lipid peroxidation (MDA), electrolyte leakage (EL) and hydrogen peroxide (H₂O₂), to a greater extent in salt-sensitive genotype. Salt-stressed plant leaves exhibited a greater activity of superoxide dismutase (SOD, EC 1.15.1.1), peroxidase (POD, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11) at 4 d after treatment (DAT), but a lower level of enzyme activity at 8 and 12 d, when compared to the control. Catalase (CAT, EC 1.11.1.6) activity was greater at 4 DAT and thereafter decreased in salt tolerant genotype relative to the control, whereas lower than the control during whole experiment period for salt-sensitive genotype. There were different patterns of five isoforms of SOD, POD and two isoforms of APX between two genotypes. Antioxidant gene expression was positively related to isoenzymatic and total enzymatic activities during 12-d salt-treated leaves of two genotypes, with a relatively higher level in salt-tolerant genotype. Thus, salt tolerance could be related to the constitutive/induced antioxidant gene, leading to more efficient enzyme stimulation and protection in perennial ryegrass.     

Identification and characterization of salt-responsive microRNAs in Populus tomentosa by high-throughput sequencing

Salt is one of the main environmental factors limiting plant growth and a better understanding of mechanisms of salt stress would aid efforts to bolster plant salt tolerance. MicroRNAs are well known for their important regulatory roles in response to abiotic stress in plants. In this study, high-throughput sequencing was employed to identify miRNAs in Populus tomentosa plantlets treated or not with salt (200 mM for 10 h). We found 141 conserved miRNAs belonging to 31 families, 29 non-conserved but previously-known miRNAs belonging to 26 families, and 17 novel miRNAs. Under salt stress, 19 miRNAs belonging to seven conserved miRNA families were significantly downregulated, and two miRNAs belonging to two conserved miRNA families were upregulated. Of seven non-conserved miRNAs with significantly altered expression, five were downregulated and two were upregulated. Furthermore, eight miRNAs were validated by qRT-PCR and their dynamic differential expressions were analyzed. In addition, 269 target genes of identified miRNAs were predicted and categorized by function. These results provide new insights into salt-responsive miRNAs in Populus.     

A novel in vitro tissue culture approach to study salt stress responses in citrus

In citrus, a major crop throughout the world, growth and yield are seriously affected by salinity. Different approaches, including agronomical, physiological and molecular methods, have been used to address this problem. In this work, an in vitro experimental system has been developed to study the toxic effect of NaCl on three citrus genotypes, avoiding the ion filter that represents the root system. To carry out the experiments, shoots were obtained from nodal segments of Cleopatra mandarin, Carrizo citrange and citrumelo CPB4475 plants growing in a greenhouse. Shoots were cultured in control or NaCl-supplemented media. After testing several salt concentrations, 60 mM NaCl was selected as saline treatment. Shoots accumulated similar levels of chloride when cultured without roots and exhibited similar leaf damage. No increases in malondialdehyde levels were observed in any genotype (as a measure of oxidative stress). Similar patterns of hormonal signalling (in terms of abscisic acid and salicylic acid contents) were exhibited in the three genotypes, despite their different tolerance under field conditions. All data together indicate that, without root system, all genotypes had the same behaviour under salt stress. The in vitro culture system has been proved as a useful tool to study biochemical processes involved in the response of citrus to salt stress.     

Comparison of vetiver root essential oils from cleansed (bacteria- and fungus-free) vs. non-cleansed (normal) vetiver plants

‘Karnataka’ and ‘Malaysia’ cultivars of vetiver (Vetiveria zizanioides (L.) Nash, =Chrysopogon zizanioides (L.) Roberty) were subjected to meristem tissue culture in order to produce plants that were bacteria- and fungi-free. Tissue cultured (“cleansed” or phytosanitary) vetiver was grown for five months in sterilized soil contained in pots, and the oil content of plants grown on the medium was compared to that of non-cleansed (normal) vetiver plants grown in unsterilized soil under the same conditions. Statistical analysis of 49 of the major oil components revealed numerous significant differences between tissue culture derived and natural plants for both genotypes.     

Salt and genotype impact on plant physiology and root proteome variations in tomato

To evaluate the genotypic variation of salt stress response in tomato, physiological analyses and a proteomic approach have been conducted in parallel on four contrasting tomato genotypes. After a 14 d period of salt stress in hydroponic conditions, the genotypes exhibited different responses in terms of plant growth, particularly root growth, foliar accumulation of Na(+), and foliar K/Na ratio. As a whole, Levovil appeared to be the most tolerant genotype while Cervil was the most sensitive one. Roma and Supermarmande exhibited intermediary behaviours. Among the 1300 protein spots reproducibly detected by two-dimensional electrophoresis, 90 exhibited significant abundance variations between samples and were submitted to mass spectrometry for identification. A common set of proteins (nine spots), up- or down-regulated by salt-stress whatever the genotype, was detected. But the impact of the tomato genotype on the proteome variations was much higher than the salt effect: 33 spots that were not variable with salt stress varied with the genotype. The remaining number of variable spots (48) exhibited combined effects of the genotype and the salt factors, putatively linked to the degrees of genotype tolerance. The carbon metabolism and energy-related proteins were mainly up-regulated by salt stress and exhibited most-tolerant versus most-sensitive abundance variations. Unexpectedly, some antioxidant and defence proteins were also down-regulated, while some proteins putatively involved in osmoprotectant synthesis and cell wall reinforcement were up-regulated by salt stress mainly in tolerant genotypes. The results showed the effect of 14 d stress on the tomato root proteome and underlined significant genotype differences, suggesting the importance of making use of genetic variability.     

Peach fruit ripening: A proteomic comparative analysis of the mesocarp of two cultivars with different flesh firmness at two ripening stages

A proteomic analysis was conducted on peach fruit mesocarp in order to better elucidate the biochemical and physiological events which characterize the transition of fruit from the “unripe” to the “ripe” phase.The first goal of the present work was to set-up a protocol suitable for improving protein extraction from peach mesocarp. The use of freeze-dried powdered tissue, together with the addition of phenol prior to the extraction with an aqueous buffer, significantly increased the protein yield and the quality of 2-DE gels. The proteomic profiles of the mesocarp from peach fruit of a non-melting flesh (NMF; ‘Oro A’) and a melting flesh (MF; ‘Bolero’) cultivar, at “unripe” and “ripe” stages as defined by some parameters typical of ripening, were then analyzed.The comparative analysis of the 2-DE gels showed that in NMF and MF peaches the relative volumes of 53 protein spots significantly changed in relation to both the ripening stage (“unripe” versus “ripe”) and/or the genetic background of the cultivar (‘Oro A’ versus ‘Bolero’).Thirty out of the 53 differently abundant spots were identified by LC-ESI-MS/MS. The analysis revealed enzymes involved in primary metabolism (e.g. C-compounds, carbohydrates, organic acids and amino acids) and in ethylene biosynthesis as well as proteins involved in secondary metabolism and responses to stress.Among these, 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) appeared to be one of the proteins with the largest change in relative abundance during the fruit transition from the pre-climacteric (“unripe”) to the climacteric (“ripe”) phase. Other proteins, such as S-adenosylmethionine synthetase and β-cyanoalanine synthase involved in ethylene metabolism, were also identified. Moreover, the changes in the relative abundances of a sucrose synthase and an α-amylase suggested differences between the two cultivars in the carbohydrate import activity of ripe fruit. The different accumulation of a few typical ROS-scavenger enzymes suggested that a higher oxidative stress occurred in MF with respect to NMF fruit. This result, together with data concerning the levels of total proteins and free amino acids and those regarding proteins involved in the maintenance of tissue integrity, was consistent with the hypothesis that the last phase of ripening in MF fruit is characterized by the appearance of a senescence status.The present study appears to define well some of the biochemical and physiological events that characterize the ripening of peach and, at the same time, provides interesting indications that could be employed in future marker assisted selection (MAS) programmes aimed to obtain MF fruits with higher ability to preserve tissue functionality maintaining for a longer time their organoleptic characteristics.     

Himantura tutul sp. nov. (Myliobatoidei: Dasyatidae), a new ocellated whipray from the tropical Indo-West Pacific,described from its cytochrome-oxidase I gene sequence

It has been previously established that the Leopard Whipray, Himantura leoparda, consists of two genetically isolated, cryptic species, provisionally designated as ‘Cluster 1’ and ‘Cluster 4’ (Arlyza et al., Mol. Phylogenet. Evol. 65 (2013) [1]). Here, we show that the two cryptic species differ by the spotting patterns on the dorsal surface of adults: Cluster-4 individuals tend to have larger-ocellated spots, which also more often have a continuous contour than Cluster-1 individuals. We show that H. leoparda's holotype has the typical larger-ocellated spot pattern, designating Cluster 4 as the actual H. leoparda. The other species (Cluster 1) is described as Himantura tutul sp. nov. on the basis of the nucleotide sequence of a 655-base pair fragment of its cytochrome-oxidase I gene (GenBank accession No. JX263335). Nucleotide synapomorphies at this locus clearly distinguish H. tutul sp. nov. from all three other valid species in the H. uarnak species complex, namely H. leoparda, H. uarnak, and H. undulata. H. tutul sp. nov. has a wide distribution in the Indo-West Pacific, from the shores of eastern Africa to the Indo-Malay archipelago. H. leoparda under its new definition has a similarly wide Indo-West Pacific distribution.     

Echinococcus granulosus sensu lato genotypes infecting humans – review of current knowledge

Genetic variability in the species group Echinococcus granulosus sensu lato is well recognised as affecting intermediate host susceptibility and other biological features of the parasites. Molecular methods have allowed discrimination of different genotypes (G1–10 and the ‘lion strain’), some of which are now considered separate species. An accumulation of genotypic analyses undertaken on parasite isolates from human cases of cystic echinococcosis provides the basis upon which an assessment is made here of the relative contribution of the different genotypes to human disease. The allocation of samples to G-numbers becomes increasingly difficult, because much more variability than previously recognised exists in the genotypic clusters G1–3 (=E. granulosus sensu stricto) and G6–10 (Echinococcus canadensis). To accommodate the heterogeneous criteria used for genotyping in the literature, we restrict ourselves to differentiate between E. granulosus sensu stricto (G1–3), Echinococcus equinus (G4), Echinococcus ortleppi (G5) and E. canadensis (G6–7, G8, G10). The genotype G1 is responsible for the great majority of human cystic echinococcosis worldwide (88.44%), has the most cosmopolitan distribution and is often associated with transmission via sheep as intermediate hosts. The closely related genotypes G6 and G7 cause a significant number of human infections (11.07%). The genotype G6 was found to be responsible for 7.34% of infections worldwide. This strain is known from Africa and Asia, where it is transmitted mainly by camels (and goats), and South America, where it appears to be mainly transmitted by goats. The G7 genotype has been responsible for 3.73% of human cases of cystic echinococcosis in eastern European countries, where the parasite is transmitted by pigs. Some of the samples (11) could not be identified with a single specific genotype belonging to E. canadensis (G6/10). Rare cases of human cystic echinococcosis have been identified as having been caused by the G5, G8 and G10 genotypes. No cases of human infection with G4 have been described. Biological differences between the species and genotypes have potential to affect the transmission dynamics of the parasite, requiring modification of methods used in disease control initiatives. Recent investigations have revealed that the protective vaccine antigen (EG95), developed for the G1 genotype, is immunologically different in the G6 genotype. Further research will be required to determine whether the current EG95 vaccine would be effective against the G6 or G7 genotypes, or whether it will be necessary, and possible, to develop genotype-specific vaccines.     

Differential responses of two Stellaria longipes ecotypes to ultraviolet-B radiation and drought stress

Enhanced ultraviolet-B (UVB) radiation and water deficit affect plant growth and development. We determined the effects of UVB and drought stress on growth parameters and chemical attributes of two ecotypes (alpine and prairie) of Stellaria longipes under controlled-environment conditions. Clonal ramets of these ecotypes were grown under three UVB levels (0, 5, and 10 kJ m⁻² d⁻¹) and exposed to two watering regimes (well watered and drought stressed) for 21 days. Compared to the alpine, the prairie ecotype was taller, had higher number of nodes, and greater leaf area and specific leaf weight (leaf dry weight: leaf area), which resulted in increased dry matter in this ecotype. Overall, ‘prairie’ was higher in total chlorophyll (Chl), but lower in Chla:b ratio, flavonoids, and ethylene, than ‘alpine’. In both ecotypes, UVB and drought stress reduced growth and dry matter, whereas UVB increased carotenoids and flavonoids. Drought stress decreased ethylene evolution. These characteristics were also determined in plants growing in the field. In the field-growing plants, ‘prairie’ had higher growth and dry matter, but lower Chla:b ratio and flavonoids, than ‘alpine’. The two ecotypes responded differentially to UVB and watering regime, as ‘prairie’ appeared to be more sensitive to UVB and drought stress than ‘alpine’.