Cytotoxic and apoptotic activities of extract of <Emphasis Type="Italic">Amaranthus</Emphasis><Emphasis Type="Italic">spinosus</Emphasis> L. in <Emphasis Type="Italic">Allium cepa</Emphasis> and human erythrocytes

The present study examined the apoptosis inducing effects of Amaranthus spinosus L. aqueous extract in Allium cepa root meristematic cells and human erythrocytes. Cytogenetic assay revealed many apoptosis inducing cytogenetic aberrations viz., cytoplasmic breakage, cytoplasmic disintegration, cytoplasmic shrinkage, receding of cytoplasm, cytoplasmic vacuolation, enucleated cell, ghost cell, nuclear vacuolation, nuclear fragmentation and nuclear disintegration. A remarkable modification of red blood cell surface morphology was observed in the result of RBC assay. The treated RBCs show membrane blebbing and shrinkage, features typical for apoptosis in nucleated cells. Significant induction of cell death was observed in treated Allium root tip cells after Evans blue staining, disclosing the membrane damage potential of the plant extract. TTC assay results in reduced mitochondrial/metabolic activity in Allium root tip cells after treatment, designating the adverse effect of plant extract on mitochondrial respiratory chain. These results confirm the apoptosis inducing potential of A. spinosus extract. Confirming the present results by further in vitro studies, it can be effectively targeted against cell proliferation during cancer treatment by inducing apoptosis. Thus from the present investigation it can be concluded that the aqueous extract of A. spinosus exhibited apoptosis induction and cytotoxic activities.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

<Emphasis Type="Italic">SnRK2</Emphasis> Homologs in <Emphasis Type="Italic">Gossypium</Emphasis> and <Emphasis Type="Italic">GhSnRK2.6</Emphasis> Improved Salt Tolerance in Transgenic Upland Cotton and <Emphasis Type="Italic">Arabidopsis</Emphasis>

SnRK2s are a large family of plant-specific protein kinases, which play important roles in multiple abiotic stress responses in various plant species. But the family in Gossypium has not been well studied. Here, we identified 13, 10, and 13 members of the SnRK2 family from Gossypium raimondii, Gossypium arboreum, and Gossypium hirsutum, respectively, and analyzed the locations of SnRK2 homologs in chromosomes based on genome data of cotton species. Phylogenetic tree analysis of SnRK2 proteins showed that these families were classified into three groups. All SnRK2 genes were comprised of nine exons and eight introns, and the exon distributions and the intron phase of homolog genes among different cotton species were analogous. Moreover, GhSnRK2.6 was overexpressed in Arabidopsis and upland cotton, respectively. Under salt treatment, overexpressed Arabidopsis could maintain higher biomass accumulation than wild-type plants, and GhSnRK2.6 overexpression in cotton exhibited higher germination rate than the control. So, the gene GhSnRK2.6 could be utilized in cotton breeding for salt tolerance.     

<Emphasis Type="Italic">Trichoderma</Emphasis> Modulates Stomatal Aperture and Leaf Transpiration Through an Abscisic Acid-Dependent Mechanism in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Trichoderma species are widespread phytostimulant fungi that act through biocontrol of root pathogens, modulation of root architecture, and improving plant adaptation to biotic and abiotic stress. With the major challenge to better understand the contribution of Trichoderma symbionts to plant adaptation to climate changes and confer stress tolerance, we investigated the potential of Trichoderma virens and Trichoderma atroviride in modulating stomatal aperture and plant transpiration. Arabidopsis wild-type (WT) seedlings and ABA-insensitive mutants, abi1-1 and abi2-1, were co-cultivated with either T. virens or T. atroviride, and stomatal aperture and water loss were determined in leaves. Arabidopsis WT seedlings inoculated with these fungal species showed both decreased stomatal aperture and reduced water loss when compared with uninoculated seedlings. This effect was absent in abi1-1 and abi2-1 mutants. T. virens and T. atroviride induced the abscisic acid (ABA) inducible marker abi4:uidA and produced ABA under standard or saline growth conditions. These results show a novel facet of Trichoderma-produced metabolites in stomatic aperture and water-use efficiency of plants.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

Distribution and habitats of <Emphasis Type="Italic">Phengaris</Emphasis> (<Emphasis Type="Italic">Maculinea</Emphasis>) butterflies and population ecology of <Emphasis Type="Italic">Phengaris</Emphasis><Emphasis Type="Italic">teleius</Emphasis> in China

Many species of the butterfly genus Phengaris are regarded as endangered in many parts of their distribution. Several species are also widely distributed across northern China. Due to land use change and overgrazing, their habitats are declining and many patches have been lost. This paper investigates the distribution and habitats of the Chinese Phengaris species (of the subgenus Maculinea). Shrub-grassland near forests seem the most frequent habitat for Phengaris, while flat open grasslands are mostly over-grazed and thus survival for Phengaris butterflies there seems difficult. Throughout Europe, P. teleius is an endangered species, while there is still no information on its status in China. To improve the knowledge on the population ecology of P. teleius, its population structure, adult behaviour and movement were studied through mark–release–recapture methods in the Qinling Mountains of Taibai County. Eight grassland patches which were potentially suitable were found in the area in 2013. In total, 480 individuals (274 females) were marked, resulting in an overall recapture rate of 16 %. The average daily population size was 44 butterflies (±23 SD) during the adult flight period. Sixty-seven percent of the females and 38 % of the males moved less than 50 m, and 17 % of recaptured females and 38 % of males moved more than 200 m. The mean movement distance was 107 ± 177 m for males and 182 ± 122 m for females. The majority of the recaptures (86 %) were made within the patches, only a few individuals (14 %) moved between patches. Due to human disturbance and destruction, all of the eight potentially suitable patches are becoming smaller and increasingly isolated, thus these populations of P. teleius may face an increasing risk of extinction, which may well be a tip of the iceberg of habitat loss and fragmentation of P. teleius in Taibai County and possibly beyond. Hence we hope our initial study of P. teleius could have positive impacts on the conservation of Phengaris butterflies in China.     

<Emphasis Type="Italic">Piriformospora indica</Emphasis>-mediated salinity tolerance in <Emphasis Type="Italic">Aloe vera</Emphasis> plantlets

Piriformospora indica, a root endophytic fungus, has been reported to promote growth of many plants under normal condition and allow the plants to survive under stress conditions. However, its impact on an important medicinal plant Aloe vera L. has not been well studied. Therefore, this study was undertaken to investigate the effect of P. indica on salinity stress tolerance of A. vera plant. P. indica inoculated and non-inoculated A. vera plantlets were subjected to four levels of salinity treatment- 0, 100, 200 and 300 mM NaCl. The salinity stress decreased the ability of the fungus to colonize roots of A. vera but the interaction of A. vera with P. indica resulted in an overall increase in plant biomass and greater shoot and root length as well as number of shoots and roots. The photosynthetic pigment (Chl a, Chl b and total Chl) and gel content were significantly higher for the fungus inoculated A. vera plantlets, at respective salinity concentrations. Furthermore, the inoculated plantlets had higher phenol, flavonoid, flavonol, aloin contents and radical scavenging activity at all salinity concentrations. The higher phenolic and flavonoid content may help the plants ameliorate oxidative stress resulting from high salinity.     

Features of expression of the <Emphasis Type="Italic">PsSst1</Emphasis> and <Emphasis Type="Italic">PsIgn1</Emphasis> genes in nodules of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) symbiotic mutants

The sequences of the PsSst1 and PsIgn1 genes of pea (Pisum sativum L.) homologous to the symbiotic LjSST1 and LjIGN1 genes of Lotus japonicus (Regel.) K. Larsen are determined. The expression level of PsSst1 and PsIgn1 genes is determined by real-time PCR in nodules of several symbiotic mutants and original lines of pea. Lines with increased (Sprint-2Fix^– (Pssym31)) and decreased (P61 (Pssym25)) expression level of both genes are revealed along with the lines characterized by changes in the expression level of only one of these genes. The revealed features of the PsSst1 and PsIgn1 expression allow us to expand the phenotypic characterization of pea symbiotic mutants. In addition, PsSst1 and PsIgn1 cDNA is sequenced in selected mutant lines, characterized by a decreased expression level of these genes in nodules, but no mutations are found.     

<Emphasis Type="Italic">PAD4</Emphasis>, <Emphasis Type="Italic">LSD1</Emphasis> and <Emphasis Type="Italic">EDS1</Emphasis> regulate drought tolerance,plant biomass production,and cell wall properties

Key message

Arabidopsis and poplar with modified PAD4, LSD1 and EDS1 genes exhibit successful growth under drought stress. The acclimatory strategies depend on cell division/cell death control and altered cell wall composition.

Abstract

The increase of plant tolerance towards environmental stresses would open much opportunity for successful plant cultivation in these areas that were previously considered as ineligible, e.g. in areas with poor irrigation. In this study, we performed functional analysis of proteins encoded by PHYTOALEXIN DEFICIENT 4 (PAD4), LESION SIMULATING DISEASE 1 (LSD1) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) genes to explain their role in drought tolerance and biomass production in two different species: Arabidopsis thaliana and Populus tremula × tremuloides. Arabidopsis mutants pad4-5, lsd1-1, eds1-1 and transgenic poplar lines PAD4-RNAi, LSD1-RNAi and ESD1-RNAi were examined in terms of different morphological and physiological parameters. Our experiments proved that Arabidopsis PAD4, LSD1 and EDS1 play an important role in survival under drought stress and regulate plant vegetative and generative growth. Biomass production and acclimatory strategies in poplar were also orchestrated via a genetic system of PAD4 and LSD1 which balanced the cell division and cell death processes. Furthermore, improved rate of cell division/cell differentiation and altered physical properties of poplar wood were the outcome of PAD4- and LSD1-dependent changes in cell wall structure and composition. Our results demonstrate that PAD4, LSD1 and EDS1 constitute a molecular hub, which integrates plant responses to water stress, vegetative biomass production and generative development. The applicable goal of our research was to generate transgenic plants with regulatory mechanism that perceives stress signals to optimize plant growth and biomass production in semi-stress field conditions.

     

Functional and ecophysiological traits of <Emphasis Type="Italic">Halimione portulacoides</Emphasis> and <Emphasis Type="Italic">Sarcocornia perennis</Emphasis> ecotypes in Mediterranean salt marshes under different tidal exposures

The present paper aims to discuss the functional and ecophysiological traits of two abundant succulent halophytes in Mediterranean salt marsh systems, considering two ecotypes differing on their tidal exposure. In Halimione portulacoides the higher root/shoot (R/S) biomass ratio verified in low inundation frequency (LF) marshes, as compared with that in high inundation frequency (HF), indicates an optimal partitioning strategy, inducing differential allocation patterns along with varying environmental factors. Conversely, Sarcocornia perennis appear to have an isometric allocation strategy, based on intrinsic biomass allocation mechanisms independent from the surrounding environmental conditions. Similarly, the photosynthetic traits appear to reflect the intrinsic ecology of the species. Sarcocornia perennis habitat adaptation to HF conditions is clearly revealed by its high photosynthetic efficiency under HF conditions. On the other hand, the cosmopolitan H. portulacoides shows no differences between ecotypes, in which concerns this trait, mostly due to the high carotenoid content of the HF ecotype, which allows this specie to counteract the stress generated during submersion. Additionally, anti-oxidant enzymatic activities revealed that S. perennis presents a high degree of stress tolerance under HF conditions, while H. portulacoides is highly adapted to LF conditions. These functional (biomass-related) and physiological traits determine the optimal habitats for H. portulacoides and S. perennis. While H. portulacoides optimal biomass partitioning, along with its efficient anti-oxidant defence system, makes this species fitter to LF conditions, S. perennis isometric allocation strategy, along with a higher photosynthetic efficiency under frequent flooding conditions, provides this species the mechanisms to efficiently colonize the intertidal habitat.     

New microsatellite markers developed from <Emphasis Type="Italic">Urochloa humidicola</Emphasis> (Poaceae) and cross amplification in different <Emphasis Type="Italic">Urochloa</Emphasis> species

Background

Urochloa humidicola is a forage grass that grows in tropical regions and is recognized for its tolerance to seasonal flooding. It is a polyploid and apomictic species with high phenotypic plasticity. As molecular tools are important in facilitating the development of new cultivars and in the classification of related species, the objectives of this study were to develop new polymorphic microsatellite markers from an enriched library constructed from U. humidicola and to evaluate their transferability to other Urochloa species.

Findings

Microsatellite sequences were identified from a previously constructed enriched library, and specific primers were designed for 40 loci. Isolated di-nucleotide repeat motifs were the most abundant followed by tetra-nucleotide repeats. Of the tested loci, 38 displayed polymorphism when screened across 34 polyploid Urochloa sp. genotypes, including 20 accessions and six hybrids of U. humidicola and two accessions each from U. brizantha, U. dictyoneura, U. decumbens and U. ruziziensis. The number of bands per Simple Sequence Repeat (SSR) locus ranged from one to 29 with a mean of 11.5 bands per locus. The mean Polymorphism Information Content (PIC) of all loci was 0.7136, and the mean Discrimination Power (DP) was 0.7873. Six loci amplified in all species tested. STRUCTURE analysis revealed six different allelic pools, and the genetic similarity values analyzed using Jaccard's coefficient ranged from 0.000 to 0.913.

Conclusions

This work reports new polymorphic microsatellite markers that will be useful for breeding programs for Urochloa humidicola and other Urochloa species as well as for genetic map development, germplasm characterization, evolutionary and taxonomic studies and marker-assisted trait selection.

     

An ATP-Binding Cassette Transporter Gene from <Emphasis Type="Italic">Cucumis sativus</Emphasis> L., <Emphasis Type="Italic">CsABC19</Emphasis>, Is Involved in Propamocarb Stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A propamocarb-responsive gene named CsABC19 was isolated from a cucumber cultivar ‘D0351’ using a homologous cloning strategy. The full-length cDNA of CsABC19 was 921 bp with a complete ORF encoding 306 amino acids. Quantitative real-time PCR analysis revealed that CsABC19 was induced in the root, stem, leaf, and fruit by propamocarb and the expression levels of CsABC19 seemed to be different in different tissues. Further functional analysis showed that CsABC19 transgenic Arabidopsis plants appeared better growth performance under propamocarb stress and lower propamocarb residues. Our findings suggest that CsABC19 plays a crucial role in plant responses to propamocarb stress and also provide new clues for the mechanism regulation of the responses to propamocarb stress in cucumber.     

Cloning <Emphasis Type="Italic">PIP</Emphasis> genes in drought-tolerant vetiver grass and responses of transgenic <Emphasis Type="Italic">VzPIP2;1</Emphasis> soybean plants to water stress

Vetiver grass [Vetiveria zizanioides (L.) Nash] displays comprehensive abiotic stress tolerance closely related to fine maintenance of plant water relation mediated by plasma membrane intrinsic proteins (PIPs). Two open reading frame sequences of PIPs (867 and 873 bp) were cloned from vetiver grass and named as VzPIP1;1 and VzPIP2;1, respectively. Expression of green fluorescent protein revealed only subcellular localization of VzPIP2;1 in the plasma membrane. Agrobacterium tumefaciens mediated transgenic (VzPIP2;1) soybean plants had a higher water content in above-ground parts under sufficient water supply through enhancing transpiration as compared to the non-transgenic plants but displayed a more severe drought injury because of a lower photosynthesis and a higher transpiration rate. However, A. rhizogenes mediated transgenic soybean plants kept a higher water content in above-ground parts by improving root water transport and kept a more effective photosynthesis under normal and drought conditions.     

Taxonomy and pathogenicity of <Emphasis Type="Italic">Leptographium</Emphasis> species associated with <Emphasis Type="Italic">Ips subelongatus</Emphasis> infestations of <Emphasis Type="Italic">Larix</Emphasis> spp. in northern China,including two new species

The larch bark beetle (Ips subelongatus), which occurs in larch plantations over a vast area of eastern Asia, infects both dying and fallen trees. When its population reaches a high density, the beetle may also infect healthy trees, resulting in tree decline and, eventually, death. Leptographium spp., in both their sexual and asexual states, are mainly associated with conifer-infesting bark beetles; some species are important tree pathogens. The aims of this study were to identify the Leptographium spp. associated with I. subelongatus infestations of Larix spp. in northern China and to examine their pathogenicity towards the tree. Morphological studies and phylogenetic approaches based on multilocus DNA sequence data (ITS2- partial r28S, partial β-tubulin, and EF-1α gene regions) showed that three Leptographium species occur in association with I. subelongatus in the areas investigated: Leptographium taigense, which is recorded in China for the first time, and two new species, namely L. innermongolicum sp. nov. and L. zhangii sp. nov. Leptographium innermongolicum is closely related to L. taigense, whereas L. zhangii belongs to the Grosmannia piceaperda species complex. The pathogenicity of these Leptographium species towards mature Larix spp. was tested by stem inoculation in forests. All inoculations only resulted in small lesions on the inner bark; therefore, the three Leptographium species were not considered to be pathogenic.