Identification of microRNAs and their targets from Populus euphratica

Populus euphratica is an ideal model system for research into the abiotic stress resistance research of woody plants. Although microRNAs have been found in poplars and have been shown to have diverse biological functions, a majority of them are genus- or specie-specific and few microRNAs have been found in P. euphratica to date. In this study, microRNA cloning and computational expressed sequence tag analysis were used to identify 72 putative miRNA sequences in P. euphratica. These sequences could be classified into 21 families, 12 of which were novel, increasing the number of known poplar microRNA families from 42 to 54. Expression analysis indicated that five P. euphratica microRNAs were induced by dehydration stress. Bioinformatics prediction showed that the 130 target genes are involved in development, resistance to stress, and other cellular processes. These results suggest several roles for miRNAs in the regulatory networks associated with the abiotic stress resistance of tree species.     

Over-expression of calcium-dependent protein kinase 13 and calreticulin interacting protein 1 confers cold tolerance on rice plants

Calcium is a ubiquitous signaling molecule and changes in cytosolic calcium concentration are involved in plant responses to various stimuli. The rice calcium-dependent protein kinase 13 (CDPK13) and calreticulin interacting protein 1 (CRTintP1) have previously been reported to be involved in cold stress response in rice. In this study, rice lines transformed with sense CDPK13 or CRTintP1 constructs were produced and used to investigate the function of these proteins. When the plants were incubated at 5°C for 3 days, leaf blades of both the sense transgenic and vector control rice plants became wilted and curled. When the plants were transferred back to non-stress conditions after cold treatment, the leaf blades died, but the sheaths remained green in the sense transgenic rice plants. Expression of CDPK13 or CRTintP1 was further examined in several rice varieties including cold-tolerant rice varieties. Accumulation of these proteins in the cold-tolerant rice variety was higher than that in rice varieties that are intermediate in their cold tolerance. To examine whether over-expression of CDPK13 and CRTintP1 would have any effect on the proteins or not, sense transgenic rice plants were analyzed using proteomics. The 2D-PAGE profiles of proteins from the vector control were compared with those of the sense transgenic rice plants. Two of the proteins that differed between these lines were calreticulins. The results suggest that CDPK13, calreticulin and CRTintP1 might be important signaling components for response to cold stress in rice.     

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.     

Effects of endophyte infection on drought stress tolerance of Lolium perenne accessions from the Mediterranean region

Perennial ryegrass (Lolium perenne) is a cool-season grass species that is often infected by the leaf-inhabiting endophyte Neotyphodium lolii. This particular endophyte is asexual and has the potential to impact host survival, growth and reproduction. The objective was to assess the potential costs or benefits of endophyte infection on drought stress tolerance of native perennial ryegrass accessions originally collected from Italy, Morocco, Tunisia, and Turkey. Sixty infected (E+) individuals from each accession were planted in a greenhouse. Half of these individuals were treated with a systemic fungicide to eliminate the endophyte (E−). For two drought periods water was withheld for 10-14 days and then allowed a one week recovery period following each. In some accessions under drought, E+ plants had more tillers, greater tiller lengths, total dry mass and green shoot mass than E− plants, suggesting a positive effect of endophyte infection on host growth. Total tiller length and the number of tillers showed significant population × treatment × infection interactions for 4 of 6 populations. This work is one of the few that documents the effects of endophyte infection for a common forage grass species from wild populations native to its distributional range. The results demonstrate that endophyte infection can help ameliorate abiotic stress such as drought and there may be a selective advantage for grasses from certain Mediterranean regions.     

Knock-out of Arabidopsis AtNHX4 gene enhances tolerance to salt stress

AtNHX4 belongs to the monovalent cation:proton antiporter-1 (CPA1) family in Arabidopsis. Several members of this family have been shown to be critical for plant responses to abiotic stress, but little is known on the biological functions of AtNHX4. Here, we provide the evidence that AtNHX4 plays important roles in Arabidopsis responses to salt stress. Expression of AtNHX4 was responsive to salt stress and abscisic acid. Experiments with CFP-AtNHX4 fusion protein indicated that AtNHX4 is vacuolar localized. The nhx4 mutant showed enhanced tolerance to salt stress, and lower Na⁺ content under high NaCl stress compared with wild-type plants. Furthermore, heterologous expression of AtNHX4 in Escherichia coli BL21 rendered the transformants hypersensitive to NaCl. Deletion of the hydrophilic C-terminus of AtNHX4 dramatically increased the hypersensitivity of transformants, indicating that AtNHX4 may function in Na⁺ homeostasis in plant cell, and its C-terminus plays a role in regulating the AtNHX4 activity.     

A salt-inducible chloroplastic monodehydroascorbate reductase from halophyte Avicennia marina confers salt stress tolerance on transgenic plants

Plant growth and productivity are adversely affected by various abiotic stress factors. In our previous study, we used Avicennia marina, a halophytic mangrove, as a model plant system for isolating genes functioning in salt stress tolerance. A large scale random EST sequencing from a salt stressed leaf tissue cDNA library of one month old A. marina plants resulted in identification of a clone showing maximum homology to Monodehydroascorbate reductase (Am-MDAR). MDAR plays a key role in regeneration of ascorbate from monodehydroascorbate for ROS scavenging. In this paper, we report the cellular localization and the ability to confer salt stress tolerance in transgenic tobacco of this salt inducible Am-MDAR. A transit peptide at the N-terminal region of Am-MDAR suggested that it encodes a chloroplastic isoform. The chloroplastic localization was confirmed by stable transformation and expression of the Am-MDAR-GFP fusion protein in tobacco. Transgenic tobacco plants overexpressing Am-MDAR survived better under conditions of salt stress compared to untransformed control plants. Assays of enzymes involved in ascorbate–glutathione cycle revealed an enhanced activity of MDAR and ascorbate peroxidase whereas the activity of dehyroascorbate reductase was reduced under salt stressed and unstressed conditions in Am-MDAR transgenic lines. The transgenic lines showed an enhanced redox state of ascorbate and reduced levels of malondialdehyde indicating its enhanced tolerance to oxidative stress. The results of our studies could be used as a starting point for genetic engineering of economically important plants tolerant to salt stress.     

Incidence of Venturia shoot blight in aspen (Populus tremuloides Michx.) varies with tree chemistry and genotype

Quaking aspen (Populus tremuloides) is a foundation tree species in North American forests, as well as a valuable source of wood and paper products. Quaking aspen exhibits substantial genetic variation within and among natural populations in phytochemical compounds that influence both interactions with herbivores and ecosystem dynamics. The potential association of these phytochemicals with disease resistance, however, is unknown. Here we present the results of a “natural experiment” in a common garden of quaking aspen genotypes infected with shoot blight (Venturia moreletii). We found that the incidence of shoot blight varied by 10-fold among aspen genotypes, and was strongly and negatively correlated with constitutive foliar concentrations of condensed tannins. Selection factors that shape the genetic and phytochemical architecture of aspen populations may thus simultaneously influence aspen resistance to pathogen attack, with consequences for individual tree fitness as well as community organization, via “extended phenotype” effects.     

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’.     

A novel cold-regulated gene from Camellia sinensis, CsCOR1, enhances salt- and dehydration-tolerance in tobacco

In present research, the full-length cDNA and the genomic sequence of a novel cold-regulated gene, CsCOR1, were isolated from Camellia sinensis L. The deduced protein CsCOR1 contains a hydrophobic N-terminus as a signal peptide and a hydrophilic C-terminal domain that is rich in glycine, arginine and proline. Two internal repetitive tridecapeptide fragments (HSVTAGRGGYNRG) exist in the middle of the C-terminal domain and the two nucleotide sequences encoding them are identical. CsCOR1 was localized in the cell walls of transgenic-tobaccos via CsCOR1::GFP fusion approach. The expression of CsCOR1 in tea leaves was enhanced dramatically by both cold- and dehydration-stress. And overexpression of CsCOR1 in transgenic-tobaccos improved obviously the tolerance to salinity and dehydration.     

Phylogenetically distinct Wolbachia gene and pseudogene sequences obtained from the African onchocerciasis vector Simulium squamosum

Wolbachia are intracellular bacteria mostly found in a diverse range of arthropods and filarial nematodes. They have been classified into seven distinct ‘supergroups’ and other lineages on the basis of molecular phylogenetics. The arthropod-infecting Wolbachia are usually regarded as reproductive parasites because they manipulate their host species’ sexing system to enhance their own spread, and this has led to their investigation as potential agents of genetic control in medical entomology. We report 12 partial Wolbachia gene sequences from: aspC, aspS, dnaA, fbpA, ftsZ, GroEL, hcpA, IDA, rpoB, rpe, TopI and wsp as well as a single ftsZ pseudogene sequence, which have all been PCR-amplified from Simulium squamosum (Diptera: Simuliidae). To our knowledge this is the first such report from Simuliidae. Uninterrupted open-reading frame sequences were obtained from all 12 genes, covering ∼6.2 kb of unique DNA sequence. Phylogenetic analyses with the different coding genes gave consistent results suggesting that the Wolbachia sequences obtained here do not derive from any of the known Wolbachia supergroups or lineages. Consistent with a unique genetic status for the S. squamosumWolbachia, the hypervariable regions of the Wolbachia-specific wsp gene were distinct from all previous records in both sequence and length. As well as potential implications for newly emerging Wolbachia-based disease control methods, the results may be relevant to some problems experienced in the laboratory colonisation of Simulium damnosum sensu lato and why it is such a diverse species complex.     

A new species of ferret-badger, Genus Melogale, from Vietnam

Ferret-badgers, genus Melogale, are distributed in the Indochinese region, Java, Bali and NE-Borneo. There are currently four species described each having very similar phenotypes. In March 2005, a living ferret-badger of a different phenotype was confiscated by rangers from Cuc Phuong National Park, Vietnam. This individual died and the carcass was not preserved. In January 2006, a newly deceased individual of the same phenotype was found at the Endangered Primate Rescue Center, Cuc Phuong National Park. Due to several different characteristics these individuals vary greatly from the current species. Thus, we describe an additional species, M. cucphuongensis sp. nov. from northern Vietnam, which occurs sympatrically with M. moschata and M. personata, but differs from both species clearly in skull morphology and other features.Based on a 423 bp-long fragment of the mitochondrial cytochrome b gene, M. cucphuongensis sp. nov. is a member of the genus Melogale and represents a sister lineage to a clade consisting of M. personata and M. moschata.     

Photosynthetic characteristics and growth performance of closely related aspen taxa: On the systematic relatedness of the Eurasian Populus tremula and the North American P. tremuloides

The European and American aspen species Populus tremula and P. tremuloides are closely related taxa with very large distribution ranges and high economic importance. Genetic and morphological data are not fully congruent with respect to the question of the systematic relatedness of these sister taxa, pointing either at separate species on the two continents or a single aggregate species with circumarctic distribution. In a replicated growth trial with 1-year-old saplings, we compared about 30 morphological (leaf size, leaf area, leaf numbers, leaf growth, leaf phenology and the ratio of leaves lost to leaves produced) and physiological traits (A_max, quantum yield, carboxylation efficiency, maximum rates of carboxylation and electron transport, leaf dark respiration, leaf conductance, leaf water potential and WUE) with the aim to obtain evidence in support of or against the one-species hypothesis and to identify key determinants of growth in the two aspen taxa.