<Emphasis Type="Italic">SulP</Emphasis>, a nuclear gene encoding a putative chloroplast-targeted sulfate permease in<Emphasis Type="Italic"> Chlamydomonas reinhardtii</Emphasis>

Genomic, proteomic, phylogenetic and evolutionary aspects of a novel gene encoding a putative chloroplast-targeted sulfate permease of prokaryotic origin in the green alga Chlamydomonas reinhardtii are described. This nuclear-encoded sulfate permease gene (SulP) contains four introns, whereas all other known chloroplast sulfate permease genes lack introns and are encoded by the chloroplast genome. The deduced amino acid sequence of the protein showed an extended N-terminus, which includes a putative chloroplast transit peptide. The mature protein contains seven transmembrane domains and two large hydrophilic loops. This novel prokaryotic-origin gene probably migrated from the chloroplast to the nuclear genome during evolution of C. reinhardtii. The SulP gene, or any of its homologues, has not been retained in vascular plants, e.g. Arabidopsis thaliana, although it is encountered in the chloroplast genome of a liverwort (Marchantia polymorpha). A comparative structural analysis and phylogenetic origin of chloroplast sulfate permeases in a variety of species is presented.     

Enhanced Expression of <Emphasis Type="Italic">AtNHX1</Emphasis>, in Transgenic Groundnut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) Improves Salt and Drought Tolerence

Salinity and drought are main threat to agriculture productivity, to avoid further losses it is necessary to improve the genetic material of crops against these stresses In this present study, AtNHX1, a vacuolar type Na⁺/H⁺ antiporter gene driven by 35S promoter was introduced into groundnut using Agrobacterium tumefaciens transformation system. The stable integration of the AtNHX1 gene was confirmed by polymerase chain reaction (PCR) and southern blot analysis. It was found that transgenic plants having AtNHX1 gene are more resistant to high concentration of salt and water deprivation than the wild type plants. Salt and proline level in the leaves of the transgenic plants were also much higher than that of wild type plants. The results showed that overexpression of AtNHX1 gene not only improved salt tolerance but also drought tolerance in transgenic groundnut. Our results suggest that these plants could be cultivated in salt and drought-affected soils.     

Functions of a hemolysin-like protein in the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

A glucose-tolerant strain of the cyanobacterium Synechocystis sp. PCC 6803, generally referred to as wild type, produces a hemolysin-like protein (HLP) located on the cell surface. To analyze the function of HLP, we constructed a mutant in which the hlp gene was disrupted. The growth rate of the mutant was reduced when the cells were stressed by treatment with CuSO₄, CdCl₂, ZnCl₂, ampicillin, kanamycin, or sorbitol in liquid medium, suggesting that HLP may increase cellular resistance to the inhibitory effects of these compounds. Uptake assays with ¹⁰⁹Cd²⁺ using the silicone–oil layer centrifugation technique revealed that both wild type and mutant cells were labeled with ¹⁰⁹Cd²⁺ within 1 min. Although the total radioactivity was much higher in the wild-type cells, ¹⁰⁹Cd²⁺ incorporation was clearly much higher in the mutant cells after adsorbed ¹⁰⁹Cd²⁺ was removed from the cell surface by washing with EDTA. These findings suggest that HLP functions as a barrier against the adsorption of toxic compounds.     

Functional characterization of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> with alterations in the <Emphasis Type="Italic">atpE</Emphasis> gene

The FUD17 strain of Chlamydomonas reinhardtii is a photosynthesis-deficient, acetate-requiring mutant with a defect in the chloroplast atpE gene, which codes for the ε subunit of the chloroplast ATP synthase. In this work, the FUD17 mutant was examined in relation to other known ATP synthase mutants as an initial step toward using this strain to generate altered versions of the atpE gene for site-directed mutagenesis of the ε subunit. The FUD17 strain grows well and is normally pigmented in the dark (heterotrophic conditions), but cannot grow autotrophically in the light, even when media are supplemented with acetate. Under heterotrophic conditions, it shows no accumulation of the ε subunit, and much lower levels of the α and β subunits of the chloroplast ATP synthase. FUD17 shows no light-dependent oxygen evolution and shows a strong, light-dependent alteration in its chlorophyll fluorescence. These results show that FUD17 possesses similar characteristics to other ATP synthase mutants and fails to express an assembled ATP synthase complex on its thylakoid membrane. A preliminary attempt at site-directed mutagenesis is described which produced a slightly truncated form of the ε subunit, which is expressed normally in the cell. This revised version was published online in June 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">Agrobacterium</Emphasis> -mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) using a heterologous bean chitinase gene

Cotton (Gossypium hirsutum L., var. Coker 312) hypocotyl explants were transformed with three strains of Agrobacterium tumefaciens, LBA4404, EHA101 and C58, each harboring the recombinant binary vector pBI121 containing the chi gene insert and neomycin phosphotransferase (nptII) gene, as selectable marker. Inoculated tissue sections were placed onto cotton co-cultivation medium. Transformed calli were selected on MS medium containing 50 mg l⁻¹ kanamycin and 200 mg l⁻¹ cepotaxime. Putative calli were subsequently regenerated into cotton plantlets expressing both the kanamycin resistance gene and βglucuronidase (gus) as a reporter gene. Polymerase chain reaction was used to confirm the integration of chi and nptII transgenes in the T₁ plants genome. Integration of chi gene into the genome of putative transgenic was further confirmed by Southern blot analysis. ‘Western’ immunoblot analysis of leaves isolated from T₀ transformants and progeny plants (T₁) revealed the presence of an immunoreactive band with MW of approximately 31 kDa in transgenic cotton lines using anti-chitinase-I polyclonal anti-serum. Untransformed control and one transgenic line did not show such an immunoreactive band. Chitinase specific activity in leaf tissues of transgenic lines was several folds greater than that of untransformed cotton. Crude leaf extracts from transgenic lines showed in vitro inhibitory activity against Verticillium dahliae.Transgenic plants currently growing in a greenhouse and will be bioassayed for improved resistance against V. dahlia the causal against of verticilliosis in cotton.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of meadow fescue (<Emphasis Type="Italic">Festuca pratensis</Emphasis> Huds.)

Meadow fescue (Festuca pratensis Huds.) is an important cool-season forage grass in Europe and Asia. We developed a protocol for producing meadow fescue transgenic plants mediated by Agrobacterium tumefaciens transformation. Embryogenic calli derived from mature embryos were transformed with A. tumefaciens strain AGL1 carrying the binary vector pDM805, coding for the phosphinothricin acetyltransferase (bar) and β-glucuronidase (uidA) genes. Bialaphos was used as the selective agent throughout all phases of tissue culture. In total, 40 independent transgenic plants were recovered from 45 bialaphos-resistant callus lines and an average transformation efficiency of 2% was achieved. The time frame from infection of embryogenic calli with Agrobacterium to transferring the transgenic plants to the greenhouse was 18 weeks. In a study of 11 BASTA-resistant transgenic lines, the uidA gene was expressed in 82% of the transgenic lines. Southern blot analysis revealed that 82% of the tested lines integrated one or two copies of the uidA gene. C. Gao and J. Liu contributed equally to the work.     

Resistance to crown gall disease in transgenic grapevine rootstocks containing truncated <Emphasis Type="Italic">virE2</Emphasis> of <Emphasis Type="Italic">Agrobacterium</Emphasis>

A truncated form of the Ti-plasmid virE2 gene from Agrobacterium tumefaciens strains C58 and A6, and A. vitis strain CG450 was transferred and expressed in somatic embryos of grapevine rootstocks 110 Richter (Vitis rupestris × V. berlandieri), 3309 Couderc (V. rupestris × V. riparia) and Teleki 5C (V. berlandieri × V. riparia) via Agrobacterium-mediated transformation to confer resistance to crown gall disease. Transformation was confirmed in 98% of the 322 lines by enzyme-linked immunosorbent assay for the neomycin phosphotransferase II protein and 97% of 295 lines by polymerase chain reaction for the truncated virE2 transgene. Southern blot analysis revealed the insertion of truncated virE2 at one to three loci in a subset of seven transgenic 110 Richter lines. In vitro resistance screening assays based on inoculations of shoot internode sections showed reduced tumorigenicity and very small galls in 23 of 154 transgenic lines. Non-transformed controls had a 100% tumorigenicity rate with very large galls. Disease resistance assay at the whole plant level in the greenhouse revealed seven transgenic lines (3 lines of 110 Richter, 2 lines of 3309 Couderc and 2 lines of Teleki 5C) were resistant to A. tumefaciens strain C58 and A. vitis strains TM4 and CG450 with a substantially reduced percentage of inoculation sites showing gall as compared to controls. No association was found between the level of resistance to crown gall disease and the source Agrobacterium strain of virE2. Taken together, our data showed that resistance to crown gall disease can be achieved by expressing a truncated form of virE2 in grapevines.     

Effect of over-expression of <Emphasis Type="Italic">Linum usitatissimum</Emphasis> PINORESINOL LARICIRESINOL REDUCTASE (<Emphasis Type="Italic">LuPLR</Emphasis>) gene in transgenic <Emphasis Type="Italic">Phyllanthus amarus</Emphasis>

Shoot tip explants of Phyllanthus amarus were cocultivated with Agrobacterium tumefaciens strain LBA 4404 carrying plasmid pCAMBIA 2301 harbouring genes coding for betaglucuronidase (gus), kanamycin (kan), and neomycin phosphotransferase II (nptII) along with a gene coding for Linum usitatissimum PINORESINOL LARICIRESINOL REDUCTASE (Lu-PLR). Transformed shoot tip explants were maintained in a Murashige and Skoog (MS) medium containing TDZ 1.54 mg l^?1, kan 50 mg l^?1 and cephotaxime 62.5 mg l^?1. The optimum medium for regeneration of multiple shoots was MS supplemented with TDZ 1.54 mg l^?1, kan 50 mg l^?1. Efficient and effective rooting of plantlets was achieved by culturing the in vitro regenerated shoots on liquid ½ MS medium containing 0.7 mg l^?1 indole 3-butyric acid (IBA) and 5 mg l^?1 kan. Rooted plants were acclimatized in the mixtures of vermiculite and soil. The transformation of kan-resistant plantlets regenerated from shoot-tip explants was confirmed by GUS and polymerase chain reaction (PCR) analysis. Southern blot and reverse transcribed PCR (RT-PCR) analysis confirmed successful integration and expression of Lu-PLR gene. Quantitative analysis of phyllanthin performed on transgenic and wild plants using high-performance liquid chromatography (HPLC) revealed that transgenic lines contained higher phyllanthin content (0.3–0.81% w/w) than wild plants (0.09% w/w). The highest yield of phyllanthin was detected in transgenic lines was up to 1.16, 1.22 and 1.23 folds higher than that of wild plant. This report highlights the transgenic approach to enhance the contents of phyllanthin and hypophyllanthin.     

Transgenic Rice Plants Expressing a Modified <Emphasis Type="Italic">cry1Ca1</Emphasis> Gene are Resistant to <Emphasis Type="Italic">Spodoptera litura</Emphasis> and <Emphasis Type="Italic">Chilo</Emphasis><Emphasis Type="Italic">suppressalis</Emphasis>

Nucleotide sequence encoding the truncated insecticidal Cry1Ca1 protein from Bacillus thuringiensis was extensively modified based on the codon usage of rice genes. The overall G + C contents of the synthetic cry1Ca1 coding sequence were raised to 65% with an additional bias of enriching for G and C ending codons as preferred by monocots. The synthetic gene was introduced into the Chinese japonica variety, Xiushui 11, by Agrobacterium-mediated transformation. Transgenic rice plants harboring this gene were highly resistant to Chilo suppressalis and Spodoptera litura larvae as revealed by insect bioassays. High levels of Cry1Ca1 protein were obtained in the leaves of transgenic rice, which were effective in achieving 100% mortality of S. litura and C. suppressalis larvae. The levels of Cry1Ca1 expression in the leaves of these transgenic plants were up to 0.34% of the total soluble proteins. The larvae of C. suppressalis and S. litura could consume a maximum of 1.89  and 4.89 mm² of transgenic leaf area whereas the consumption of non-transgenic leaves by these larvae was significantly higher; 58.33 and 61.22 mm², respectively. Analysis of R₁ transgenic plants indicated that the cry1Ca1 was inherited by the progeny plants and provided complete protection against C. suppressalis and S. litura larvae.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Lotus tenuis</Emphasis> and regeneration of transgenic lines

A protocol for the production of transgenic plants was developed for Lotus tenuis via Agrobacterium-mediated transformation of leaf segments. The explants were co-cultivated (for 3 days) with an A. tumefaciens strain harbouring either the binary vector pBi RD29A:oat arginine decarboxylase (ADC) or pBi RD29A:glucuronidase (GUS), which carries the neomycin phosphotransferase II (nptII) gene in the T-DNA region. Following co-cultivation, the explants were cultured in Murashige and Skoog medium supplemented with naphthalenacetic acid (NAA) and benzyladenine (BA) and containing kanamycin (30 μg ml⁻¹) and cefotaxime (400 μg ml⁻¹) for 45 days. The explants were subcultured several times (at 2-week intervals) to maintain the selection pressure during the entire period. About 40% of the explants inoculated with the pBiRD29:ADC strain produced eight to ten adventitious shoots per responsive explant through a direct system of regeneration, whereas 69% of the explants inoculated with the pBi RD29A:GUS strain produced 13–15 adventitious shoots per responsive explant. The selected transgenic lines were identified by PCR and Southern blot analysis. Three ADC transgenic lines were obtained from 30 infected explants, whereas 29 GUS transgenic lines were obtained from 160 explants, corresponding to a transformation efficiency of 10 and 18.1%, respectively. More than 90% of the in vitro plantlets were successfully transferred to the soil. The increase in the activity of arginine decarboxylase from stressed ADC- Lt19 lines was accompanied by a significant rise in the putrescine level. The GUS transgenic line driven by the RD29A promoter showed strong signals of osmotic stress in the leaves and stem tissues. All of the transgenic plants obtained exhibited the same phenotype as the untransformed controls under non-stress conditions, and the stability of the gene introduced into the cloned materials was established.     

Cyanobacteria MT gene <Emphasis Type="Italic">SmtA</Emphasis> enhance zinc tolerance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Zinc is essential but toxic in excess. A bacterial metallothionein, SmtA from Synechococcus PCC 7942, has high affinity for Zn²⁺ and the intracellular exclusively handling of Zn²⁺. In this study, we report a functional analysis of SmtA in Arabidopsis thaliana and its response to zinc stress. After high zinc stress, the transgenic plants over-expressing SmtA showed higher survival rate than the wild type. We also found that over-expression of SmtA in Arabidopsis increased the activities of SOD and POD, and enhanced the tolerance to zinc stress. Together, our results indicate that SmtA may play an important role in the response to zinc stress in Arabidopsis.     

Expression of CoQ10-producing <Emphasis Type="Italic">ddsA</Emphasis> transgene by efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Panicum meyerianum</Emphasis>

Panicum meyerianum Nees is a wild relative of Panicum maximum Jacq. (guinea grass), which is an important warm-season forage grass and biomass crop. We investigated the conditions that maximized the transformation efficiency of P. meyerianum by Agrobacterium infection by monitoring the expression of the β-glucuronidase (GUS) gene. The highest activities of GUS in calli were achieved by the co-cultivation of plants with Agrobacterium at 28°C for 6 days. We transferred the ddsA gene, which encodes decaprenyl diphosphate synthase and is required for coenzyme Q10 (CoQ10) synthesis, into P. meyerianum by using our optimized co-cultivation procedure for transformation. We confirmed by PCR and DNA gel blot hybridization that all hygromycin-resistant plants retained stable insertion of the hpt and ddsA genes. We also demonstrated strong expression of S14:DdsA protein in the leaves of transgenic P. meyerianum. Furthermore, we showed that transgenic P. meyerianum produced CoQ10 at levels 11–20 times higher than that of non-transformants. By comparison, the CoQ9 level in transgenic plants was dramatically reduced. This is the first report of efficient Agrobacterium-mediated transfer of a foreign gene into the warm-season grass P. meyerianum.     

Production of transgenic <Emphasis Type="Italic">Pinus armandii</Emphasis> plants harbouring <Emphasis Type="Italic">btCry</Emphasis>III(<Emphasis Type="Italic">A</Emphasis>) gene

A synthetic chimeric gene SbtCryIII(A) encoding the insecticidal protein btCryIII(A), was transformed into Pinus armandii embryos and embryogenic calli using Agrobacterium tumefaciens. Polymerase chain reaction and genomic DNA Southern blot analysis showed that the SbtCryIII(A) gene was integrated into the genome of transgenic Pinus armandii plants, and Northern blot analysis indicated that the SbtCryIII(A) gene was transcribed.     

Heterologous expression of <Emphasis Type="Italic">ApGSMT2</Emphasis> and <Emphasis Type="Italic">ApDMT2</Emphasis> genes from <Emphasis Type="Italic">Aphanothece halophytica</Emphasis> enhanced drought tolerance in transgenic tobacco

The glycine-methylation biosynthetic pathway of glycinebetaine (GB) has been investigated, but only a few studies on GB accumulation in transgenic higher plants have utilized this pathway. In this study, two methyltransferase genes named ApGSMT2 and ApDMT2, encoding proteins catalyzing GB biosynthesis from glycine, were cloned from a relative strain of Aphanothece halophytica. The potential roles of ApGSMT2 and ApDMT2 in GB synthesis were first examined in transgenic Escherichia coli, which had increased levels of GB and improved salt tolerance. Then ApGSMT2 and ApDMT2 were transferred into tobacco. Compared with transgenic tobacco expressing betA, transgenic tobacco co-expressing ApGSMT2 and ApDMT2 accumulated more GB and exhibited enhanced drought resistance with better germination performance, higher relative water content, less cell membrane damage and better photosynthetic capacity under drought stress. We concluded that the ApGSMT2 and ApDMT2 genes cloned in this study will be very useful for engineering GB-accumulating transgenic plants with enhanced drought resistance.     

Heterologous expression of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">NPR1</Emphasis> (<Emphasis Type="Italic">AtNPR1</Emphasis>) enhances oxidative stress tolerance in transgenic tobacco plants

In Arabidopsis, NPR1 (non-expressor of pathogenesis related genes 1, AtNPR1) functions downstream of salicylic acid (SA) and modulates the SA mediated systemic acquired resistance. It is also involved in a cross talk with the jasmonate pathway that is essential for resistance against herbivores and necrotrophic pathogens. Overexpression of AtNPR1 in transgenic plants resulted in enhanced disease resistance. Recently, tobacco transgenic plants expressing AtNPR1 were shown to be tolerant to the early instars of Spodoptera litura (Meur et al., Physiol Plant 133:765–775, 2008). In this communication, we show that the heterologous expression of AtNPR1 in tobacco has also enhanced the oxidative stress tolerance. The transgenic plants exhibited enhanced tolerance to the treatment with methyl viologen. This tolerance was associated with the constitutive upregulation of PR1, PR2 (glucanase), PR5 (thaumatin like protein), ascorbate peroxidase (APX) and Cu²⁺/Zn²⁺ superoxide dismutase (SOD). This is the first demonstration of the novel function of heterologous expression of AtNPR1 in oxidative stress tolerance in transgenic tobacco.     

Highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of celery (<Emphasis Type="Italic">Apium graveolens</Emphasis> L.) through somatic embryogenesis

A protocol was developed for rapid and efficient production of transgenic celery plants via somatic embryo regeneration from Agrobacterium tumefaciens- inoculated leaf sections, cotyledons and hypocotyls. These explants were excised from in vitro seedlings of the cvs. XP166 and XP85 and inoculated with A. tumefaciens strain EHA105 containing the binary vector pBISN1. PBISN1 has the neomycin phosphotransferase gene (nptII) and an intron interrupted β-glucuronidase (GUS) reporter gene (gusA). Co-cultivation was carried out for 4 d in the dark on callus induction medium (CIM): Gamborg B5 + 2.79 μM kinetin + 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented with 100 μM acetosyringone. Embryogenic calluses resistant to kanamycin (Km) were then recovered on CIM + 25 mg l⁻¹ Km + 250 mg l⁻¹ timentin after 12 weeks. Subsequently, a large number of Km-resistant and GUS-positive transformants, tens to hundreds per explant were regenerated via somatic embryogenesis on Gamborg B5 + 4.92 μM 6 (γ,γ-dimethylallylamino)-purine (2iP) + 1.93 μM α-naphthaleneacetic acid (NAA) + 25 mg l⁻¹ Km + 250 mg l⁻¹ timentin after 8 weeks. Using this protocol, the transformation frequency was 5.0% and 5.0% for leaf sections, 17.8% and 18.3% for cotyledons, and 15.9% and 16.7% for hypocotyl explants of cvs. XP85 and XP166, respectively. Stable integration of the model transgenes with 1–3 copy numbers was confirmed in all ten randomly selected transgenic events by Southern blot analysis of gusA. Progeny analysis by histochemical GUS assay showed stable Mendelian inheritance of the transgenes. Thus, A. tumefaciens-mediated transformation of cotyledons or hypocotyls provides an effective and reproducible protocol for large-scale production of transgenic celery plants.     

Efficient expression of epidermal growth factor in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> CC400

The application of the green alga Chlamydomonas reinhardtii as a bioreactor is not adequate because of the difficulties caused by efficiency expressing foreign genes. To improve this efficiency a plasmid containing the epidermal growth factor (EGF) gene and a bleomycin resistance gene (ble) was constructed. We amplified the EGF gene according to the codon usage of C. reinhardtii. The vector carrying 2 expression cassettes for EGF gene and ble gene was constructed by adding rbc promoter and rbc terminator. Transformants, selected on Tris-acetate-phosphate medium containing 15 mg/L bleomycin, were screened by PCR and confirmed by Southern blotting, which showed that 3 transgenic C. reinhardtii cells contained only one copy of EGF gene integrated in different 3 sites of C. reinhardtii CC400 genome. Then EGF protein content of 3 transformants was determined by EGF precoated ELISA, indicating that EGF gene was first expressed, although at a low level, in algal cells. The presented study, as an example for expressing heterologous gene in green alga, provided feasibility to improve the efficiency of transformation of C. reinhardtii.     

<Emphasis Type="Italic">In vivo</Emphasis> fluorescence observation of parasporal inclusion formation in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A recombinant gene expressing a Cry1Ac-GFP fusion protein with a molecular mass of approximately 160 kD was constructed to investigate the expression of cry1Ac, the localization of its gene product Cry1Ac, and its role in crystal development in Bacillus thuringiensis. The cry1Ac-gfp fusion gene under the control of the cry1Ac promoter was cloned into the plasmid pHT304, and this construct was designated pHTcry1Ac-gfp. pHTcry1Ac-gfp was transformed into the crystal-negative strain, HD-73 cry⁻, and the resulting strain was named HD-73⁻(pHTcry1Ac-gfp). The gfp gene was then inserted into the large HD-73 endogenous plasmid pHT73 and fused with the 3′ terminal of the cry1Ac gene by homologous recombination, yielding HD-73Φ(cry1Ac-gfp)3534. Laser confocal microscopy and Western blot analyses showed for the first time that the Cry1Ac-GFP fusion proteins in both HD-73⁻(pHTcry1Ac-gfp) and HD-73Φ(cry1Ac-gfp)3534 were produced during asymmetric septum formation. Surprisingly, the Cry1Ac-GFP fusion protein showed polarity and was located near the septa in both strains. There was no significant difference between Cry1Ac-GFP and Cry1Ac in their toxicity to Plutella xylostella larvae.