Overexpression of <Emphasis Type="Italic">AtHDG11</Emphasis> enhanced drought tolerance in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Drought is one of the major abiotic stresses restricting the yield of wheat (Triticum aestivum L.). Breeding wheat varieties with drought tolerance is an effective and durable way to fight against drought. Here we reported introduction of AtHDG11 into wheat via Agrobacterium-mediated transformation and analyzed the morphological and physiological characteristics of T2 generation transgenic lines under drought stress. With drought treatment for 30 days, transgenic plants showed significantly improved drought tolerance. Compared with controls, the transgenic lines displayed lower stomatal density, lower water loss rate, more proline accumulation and increased activities of catalase and superoxide dismutase. Without irrigation after booting stage, the photosynthetic parameters, such as net photosynthesis rate, water use efficiency and efficiency of excitation energy, were increased in transgenic lines, while transpiration rate was decreased. Moreover, the kernel yield of transgenic lines was also improved under drought condition. Taken together, our data demonstrate that AtHDG11 has great potential in genetic improvement of drought tolerance of wheat.     

Physiological responses of <Emphasis Type="Italic">Lupinus luteus</Emphasis> to different copper concentrations

Yellow lupin (Lupinus luteus L.) plants were grown in hydroponic solution for 15 d under different copper concentrations (0.1, 0.5, 1.0, 10, 25 and 50 μM). With increasing Cu concentration total biomass was not affected, leaf area slightly decreased, while chlorophyll content decreased considerably. Cu content increased significantly both in roots and in leaves, but the contents of other ions were only slightly affected at the highest Cu concentration (Mn content decreased both in roots and in leaves, P content decreased only in leaves and Zn content increased in roots). Superoxide dismutase (SOD) activity increased up to day 7 after copper application. Peroxidase (GPOD) and polyphenol oxidase (PPO) activities also increased, while catalase (CAT) activity remained constant.     

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">INFLORESCENCE DEFICIENT IN ABSCISSION-like</Emphasis> is an abscission-associated and phytohormone-regulated gene in flower separation of <Emphasis Type="Italic">Lupinus luteus</Emphasis>

Abscission is a natural process that occurs to facilitate shedding of no longer needed organs, but on the other hand, can be triggered by certain environmental conditions, e.g. biotic or abiotic stresses. Regardless of the stimuli, organ shedding takes place specifically at the abscission zone (AZ). A signaling pathway that controls this process in Arabidopsis thaliana from ligand to receptors has been proposed. However, knowledge concerning the influence of plant hormones on these molecular elements still remains enigmatic. Excessive and premature flower abscission in the crop species Lupinus luteus L. is a process of substantial interest to the agricultural industry, as it can affect yield. Our strategy combined molecular studies, comprehensive ultrastructural and histological analysis, as well as exogenous hormone treatment to describe the contribution of the Lupinus IDA-like gene in flower abscission. In the AZ of the naturally abscised flowers, the differentiation of morphologically distinct cells characterized by progressive degradation processes was accompanied by LlIDL mRNA accumulation. A similar effect was observed following early steps of AZ activation and after abscisic acid or ethylene treatments. These phytohormones, previously pointed out as key stimulators of flower separation, altered the temporal expression pattern of LlIDL. Exogenous EPIP peptide synthesized on the basis of LlIDL sequence, significantly increased flower abortion rate, which indicates that this motif governs protein activity. In conclusion, our data provide new evidence for LlIDA involvement in both the early and late events of flower abscission supported by detailed spatiotemporal characterization of AZ cell structure and ultrastructure.     

Main centers of nitrate and nitrite reduction in young and nodulated yellow lupine (<Emphasis Type="Italic">Lupinus luteus</Emphasis>)

Nitrate and nitrite reduction centers in non-nodulated and symbiotic yellow lupine were analyzed. In young seedlings, nitrate was exclusively accumulated in roots, which also was shown as the main nitrate reduction center. In contrast, leaves were shown to play a key role in nitrite reduction. A similar distribution of nitrate reductase (NR) and nitrite reductase was found in nodulated plants. However, in field conditions characterized by low nitrate content, a disproportionately high level of NR activity in nodules was also observed during all stages of symbiotic growth. This feature was confirmed in nitrate-fed hydroponic cultures. Nodule NR activity was one order of magnitude higher than in roots, in spite of the small stored nitrate pool found inside nodules. This suggests that nodule NR activity had been induced not by nitrate itself but indirectly. Since bacteroids were shown to be responsible for the vast majority of nodule NR activity, the plausible explanation of this effect seems to be a dissimilatory nature of rhizobial NR. Considering that environmental nitrate could cause hypoxia inside nodules, this is the proposed way of the observed nodule NR induction.     

Heterologous expression of <Emphasis Type="Italic">PDH47</Emphasis> confers drought tolerance in <Emphasis Type="Italic">indica</Emphasis> rice

Jerusalem artichoke (Helianthus tuberosus L.) cultivars are conserved in genebanks for use in breeding and horticultural research programs. Jerusalem artichoke collections are particularly vulnerable to environmental and biological threats because they are often maintained in the field. These field collections could be securely conserved in genebanks if improved cryopreservation methods were available. This work used four Jersualem artichoke cultivars (‘Shudi’, ‘M6’, ‘Stampede’, and ‘Relikt’) to improve upon an existing procedure. Four steps were optimized and the resulting procedure is as follows: preculture excised shoot tips (2–3 mm) in liquid MS medium supplemented with 0.4 M sucrose for 3 days, osmoprotect shoot tips in loading solution for 30 min, dehydrate with plant vitrification solution 2 for 15 min before rapid cooling in liquid nitrogen, store in liquid nitrogen, rapidly rewarm in MS liquid medium containing 1.2 M sucrose, and recover on MS medium supplemented with 0.1 mg L^?1 GA₃ for 3–5 days in the dark and then on the same medium for 4–6 weeks in the light (14 h light/10 h dark). After cryopreservation, Jerusalem artichoke cultivar ‘Shudi’ had the highest survival (93%) and regrowth (83%) percentages. Cultivars ‘M6’, ‘Stampede’, and ‘Relikt’ achieved survival and regrowth percentages ranging from 44 to 72%, and 37–53%, respectively. No genetic changes, as assessed by using simple sequence repeat markers, were detected in plants regenerated after LN exposure in Jerusalem artichoke cultivar ‘Shudi’. Differential scanning calorimetry analyses were used to investigate the thermal activities of the tissues during the cryopreservation process and it was determined that loading with 2.0 M sucrose and 0.4 M sucrose dehydrated the shoot tips prior to treatment with PVS2. Histological observations revealed that the optimized droplet vitrification protocol caused minimal cellular damage within the meristem cells of the shoot tips.     

Factors affecting <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Hybanthus</Emphasis><Emphasis Type="Italic">enneaspermus</Emphasis> (L.) F. Muell.

Agrobacterium tumefaciens-mediated transformation system was established for Hybanthus enneaspermus using leaf explants with the strain LBA4404 harbouring pCAMBIA 2301 carrying the nptII and gusA genes. Sensitivity of leaf explants to kanamycin was standardized (100 mg/l) for screening the transgenic plants. Transformation parameters (OD, virulence inducer, infection time, co-cultivation period, bactericidal antibiotics, etc.) influencing the gene transfer and integration were assessed in the present investigation. Fourteen-day pre-cultured explants were subjected with Agrobacterium strain LBA4404. Optimized parameters such as culture density of 0.5 OD₆₀₀, infection time of 6 min, AS concentration of 150 µM with 3 days co-cultivation revealed maximum transformation efficiency based on GUS expression assay. The presence of gusA in transgenics was confirmed by polymerase chain reaction and Southern blotting analysis. The present transformation experiment yielded 20 shoots/explant with higher transformation efficiency (28 %). The protocol could be used to introduce genes for trait improvement as well as for altering metabolic pathway for secondary metabolites production.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated transformation of gypsophila (<Emphasis Type="Italic">Gypsophila paniculata</Emphasis> L.)

As a major contributor to the flower market, Gypsophila paniculata is an important target for the breeding of new varieties. However, gypsophila breeding is strongly hampered by the sterility of this species’ genotypes and the lack of a genetic-transformation procedure for this genus. Here we describe the establishment of a transformation procedure for gypsophila (Gypsophila paniculata L.) based on Agrobacterium inoculation of highly regenerative stem segments. The transformation procedure employs stem explants derived from GA₃-pretreated mother plants and a two-step selection scheme. The GA₃ treatment was crucial for obtaining high gene-transfer frequencies (75–90% GUS-expressing explants out of total inoculated explants), as shown using three different gypsophila varieties. An overall transformation efficiency of five GUS-expressing shoots per 100 stem explants was demonstrated for cv. Arbel. The applicability of the transformation system to gypsophila was further reinforced by the generation of transgenic plants expressing Agrobacterium rhizogenes rolC driven by a CaMV 35S promoter. Transgenic gypsophila plantlets exhibited extensive rooting and branching, traits that could be beneficial to the ornamental industry.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

High-efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation in black gram (<Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper)

In vitro culture and genetic transformation of black gram are difficult due to its recalcitrant nature. Establishment of gene transfer procedure is a prerequisite to develop transgenic plants of black gram in a shorter period. Therefore, genetic transformation was performed to optimize the factors influencing transformation efficiency through Agrobacterium tumefaciens-mediated in planta transformation using EHA 105 strain harbouring reporter gene, bar, and selectable marker, gfp-gus, in sprouted half-seed explants of black gram. Several parameters, such as co-cultivation, acetosyringone concentration, exposure time to sonication, and vacuum infiltration influencing in planta transformation, have been evaluated in this study. The half-seed explants when sonicated for 3 min and vacuum infiltered for 2 min at 100 mm of Hg in the presence of A. tumefaciens (pCAMBIA1304– bar) suspensions and incubated for 3 days co-cultivation in MS medium with 100 µM acetosyringone showed maximum transformation efficiency (46 %). The putative transformants were selected by inoculating co-cultivated seeds in BASTA^® (4 mg l^?1) containing MS medium followed by BASTA^® foliar spray on 15-day-old black gram plants (35 mg l^?1) in green house, and the transgene integration was confirmed by biochemical assay (GUS), Polymerase chain reaction, Dot-blot, and Southern hybridisation analyses.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">Micrococcus luteus</Emphasis> - Survival in Amber

A growing body of evidence now supports the isolation of microorganisms from ancient materials. However, questions about the stringency of extraction methods and the genetic relatedness of isolated organisms to their closest living relatives continue to challenge the authenticity of these ancient life forms. Previous studies have successfully isolated a number of spore-forming bacteria from organic and inorganic deposits of considerable age whose survival is explained by their ability to enter suspended animation for extended periods of time. However, despite a number of putative reports, the isolation of non-spore-forming bacteria and an explanation for their survival have remained enigmatic. Here we describe the isolation of non-spore-forming cocci from a 120-million-year-old block of amber, which by genetic, morphological, and biochemical analyses are identified as belonging to the bacterial species Micrococcus luteus. Although comparison of 16S rRNA sequences from the ancient isolates with their modern counterparts is unable to confirm the precise age of these bacteria, we demonstrate, using complementary molecular and cell biological techniques, evidence supporting the view that these (and related modern members of the genus) have numerous adaptations for survival in extreme, nutrient-poor environments, traits that will assist in this bacterias persistence and dispersal in the environment. The bacterias ability to utilize succinic acid and process terpine-related compounds, both major components of natural amber, support its survival in this oligotrophic environment.     

<Emphasis Type="Italic">S</Emphasis>-Allele characterization in self-incompatible pear (<Emphasis Type="Italic">Pyrus communis</Emphasis> L.)

Gametophytic self-incompatibility, a natural mechanism occurring in pear and other fruit-tree species, is usually controlled by the S-locus with allelic variants ( S1, S2, Sn). Recently, biochemical and molecular tools have determined the S-genotype of cultivars in various species. The present study determined the S-locus composition of ten European pear cultivars via S-PCR molecular assay, thereby obviating time-consuming fieldwork whose results are often ambiguous because of environmental effects. To verify the S-PCR assay, two putative S-allele DNA fragments of Japanese pear were isolated; their sequences proved to be identical to those reported in the databank. Six S-allele fragments of European pear were then sequenced. While field data confirmed the molecular results, fully and half-compatible field crosses were not distinguishable.     

High-resolution mapping of the <Emphasis Type="Italic">Alp</Emphasis> locus and identification of a candidate gene <Emphasis Type="Italic">HvMATE</Emphasis> controlling aluminium tolerance in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Aluminium (Al) tolerance in barley is conditioned by the Alp locus on the long arm of chromosome 4H, which is associated with Al-activated release of citrate from roots. We developed a high-resolution map of the Alp locus using 132 doubled haploid (DH) lines from a cross between Dayton (Al-tolerant) and Zhepi 2 (Al-sensitive) and 2,070 F₂ individuals from a cross between Dayton and Gairdner (Al-sensitive). The Al-activated efflux of citrate from the root apices of Al-tolerant Dayton was 10-fold greater than from the Al-sensitive parents Zhepi 2 and Gairdner. A suite of markers (ABG715, Bmag353, GBM1071, GWM165, HvMATE and HvGABP) exhibited complete linkage with the Alp locus in the DH population accounting 72% of the variation for Al tolerance evaluated as relative root elongation. These markers were used to map this genomic region in the Dayton/Gairdner population in more detail. Flanking markers HvGABP and ABG715 delineated the Alp locus to a 0.2 cM interval. Since the HvMATE marker was not polymorphic in the Dayton/Gairdner population we instead investigated the expression of the HvMATE gene. Relative expression of the HvMATE gene was 30-fold greater in Dayton than Gardiner. Furthermore, HvMATE expression in the F_2:3 families tested, including all the informative recombinant lines identified between HvGABP and ABG715 was significantly correlated with Al tolerance and Al-activated citrate efflux. These results identify HvMATE, a gene encoding a multidrug and toxic compound extrusion protein, as a candidate controlling Al tolerance in barley.     

A new <Emphasis Type="Italic">Em</Emphasis>-like protein from <Emphasis Type="Italic">Lactuca sativa</Emphasis>, <Emphasis Type="Italic">LsEm1</Emphasis>, enhances drought and salt stress tolerance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and rice

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.