A non-toxic pokeweed antiviral protein mutant inhibits pathogen infection via a novel salicylic acid-independent pathway

Pokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from Phytolacca americana, is characterized by its ability to depurinate the sarcin/ricin (S/R) loop of the large rRNA of prokaryotic and eukaryotic ribosomes. In this study, we present evidence that PAP is associated with ribosomes and depurinates tobacco ribosomes in vivo by removing more than one adenine and a guanine. A mutant of pokeweed antiviral protein, PAPn, which has a single amino acid substitution (G75D), did not bind ribosomes efficiently, indicating that Gly-75 in the N-terminal domain is critical for the binding of PAP to ribosomes. PAPn did not depurinate ribosomes and was non-toxic when expressed in transgenic tobacco plants. Unlike wild-type PAP and a C-terminal deletion mutant, transgenic plants expressing PAPn did not have elevated levels of acidic pathogenesis-related (PR) proteins. PAPn, like other forms of PAP, did not trigger production of salicylic acid (SA) in transgenic plants. Expression of the basic PR proteins, the wound-inducible protein kinase and protease inhibitor II, was induced in PAPn-expressing transgenic plants and these plants were resistant to viral and fungal infection. These results demonstrate that PAPn activates a particular SA-independent, stress-associated signal transduction pathway and confers pathogen resistance in the absence of ribosome binding, rRNA depurination and acidic PR protein production.     

Response of transgenic rape plants bearing the <Emphasis Type="Italic">Osmyb4</Emphasis> gene from rice encoding a trans-factor to low above-zero temperature

Accumulation of soluble sugars (sucrose, fructose, and glucose), proline, phenols (total phenols and flavonoids), and antocyanins during adaptation to low-temperature stress (4°C) of two lines of spring rape (Brassica napus L., cv. Westar) characterized by weak (Bn-1) and strong (Bn-3) expression of the Osmyb4 transgene was studied. Vegetatively propagated transgenic and wild-type plants were grown in the hydroponic culture at 24°C; at the stage of 5–6 leaves, plants were exposed to 4°C for 5 days and then returned to the optimum temperature of 24°C for recovery. Transgenic plants were established to manifest improved cold and frost tolerance, which was evident from more active biomass accumulation at 4°C as compared with wild-type plants and from sustaining their viability after 2-day-long exposure to −6°C. Determination of MDA content showed that one of the reasons of their improved cold tolerance was their capability of maintaining oxidative homeostasis under low-temperature stress. This suggestion is supported by intense accumulation of phenols and antocyanins, manifesting pronounced antioxidant effects, by transgenic plants during their cold adaptation. Thus, during 2–5 days of plant exposure to 4°C, in transgenic plants the total content of phenols increased by 2.6–3.7 times, flavonoids — by 3.7–4.7 times, and antocyanins — by 3.5–5.3 times as compared with control plants growing at 24°C. Transgenic Bn-3 plants with strong expression of the Osmyb4 gene accumulated phenols and antocyanins at 4°C more actively than Bn-1 plants characterized by weak expression of this gene. Transgenic rape plants subjected to cold stress accumulated more proline, manifesting stress-protection effects, and lesser accumulation of soluble sugars. Before the beginning of experiment, the content of soluble sugars was approximately similar in wild-type plants and transgenic lines; at 4°C their level in transgenic plants was substantially lower than in control plants. As distinct from the process of cold adaptation, during recovery, the content of all tested stress-protection compounds dropped sharply. The results obtained indicate that active expression of the Osmyb4 gene from rice in the rape plants was accompanied not only by accumulation of compatible osmolytes but also by biosynthesis of antioxidants of phenolic nature.     

Improving antioxidant activity in transgenic <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> plants via overexpression of the γ-tocopherol methyltransferase (γ-<Emphasis Type="Italic">tmt)</Emphasis> gene

Codonopsis lanceolata Trautv (Companulaceae) is a folk medicine in Korea. To shift the content of tocopherol and enhance its antioxidant properties, we overexpressed the γ-tocopherol methyltransferase (γ-tmt) gene in C. lanceolata. The antioxidant activity of methanolic crude extracts of the transgenic plants was compared to that of control plants using the 1,1-diphenyl-2-picrylhydrazyl radical scavenging method, with α-tocopherol and butylated hydroxy toluene as standard antioxidants. The antioxidant activity of the leaf and root extracts of transgenic plants was higher (IC ₅₀ 12–17.33 and 408–524 μg/ml, respectively) than that of control plant leaf and root extracts (18 and 529 μg/ml, respectively). High-performance liquid chromatography analysis of phenolic compounds confirmed an increase in the levels of 12 major phenolic acids and flavonoids in the leaf and root extracts of transgenic plants compared to control plants. We also found that the rate of photosynthesis was 48% higher in transgenic plants than in control plants. Based on these results, we suggest that increases in the α-tocopherol level in transgenic C. lanceolata plants may result in increases in the photosynthetic performance and antioxidant metabolism of these plants.     

Expression of the rice cytoplasmic cysteine synthase gene in tobacco reduces ozone-induced damage

Cysteine serves as a precursor for the synthesis of various sulfur-containing metabolites, and the cysteine synthase (CS) gene plays a central role in the sulfur cycle in nature. In the present study, rcs1, a cytosolic CS gene of rice, was introduced into the genome of tobacco (Nicotiana tabacum). The tolerance of wild-type tobacco plants as well as of the resulting transgenic tobacco plants overexpressing the rcs1 gene to toxic levels of ozone (O₃, 0.15 μ mol⁻¹) was measured after various lengths of exposure. Leaf lesions in plants exposed for 2 weeks to O₃ were more prevalent in the leaves of the wild-type plants than in those of the transgenic tobacco plants. Transgenic tobacco plants showed a higher growth rate and a higher chlorophyll content than the wild-type plants. Cysteine synthase activity and cysteine and glutathione contents were higher in transgenic plants than in wild-type plants irrespective of the length of the O₃ treatment. Our results indicate that the CS gene plays a role in the protection of the plant against toxic O₃ gas, probably through the mechanism of an over-accumulation of such sulfur-rich antioxidants as cysteine and glutathione.     

Transgenic Plants of Creeping Bent Grass Harboring the Stress Inducible Gene, 9-cis-epoxycarotenoid dioxygenase, are Highly Tolerant to Drought and NaCl Stress

Transgenic lines of creeping bent grass were generated by Agrobacterium-mediated transformation with the VuNCED1 which was cloned from cow pea has a homology to 9-cis-epoxycarotenoid dioxygenase, which is supposed to be involved in abscisic acid (ABA) biosynthesis. ABA, a cleavage product of carotenoids, is involved in stress responses in plants. The limiting step of ABA biosynthesis in plants is presumably the cleavage of 9-cis-epoxycarotenoids, the first committed step of ABA biosynthesis. Molecular analyses of transgenic lines as performed by Southern hybridization genomic DNA-PCR revealed integration of the VuNCED1. Challenge studies performed with transgenic plants by exposure to salt stress (up to 10 dS m⁻¹) and water stress (up to 75%) for 10 weeks, revealed that more than 50% of the transgenic plants could survive NaCl and drought stress whereas wild-type was not. ABA levels were measured under drought and normal conditions, endogenous ABA was dramatically increased by drought and NaCl stress in transgenic plants. These results indicate that it is possible to manipulate ABA levels in plants by over expressing the key regulatory gene in ABA biosynthesis and that stress tolerance can be improved by increasing ABA levels. Chenna Reddy Aswath and Sun Hyung Kim - First two authors contributed equally to this work     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of embryogenic cultures and plant regeneration in <Emphasis Type="Italic">Vitis rotundifolia</Emphasis> Michx. (muscadine grape)

A method to produce transgenic plants of Vitis rotundifolia was developed. Embryogenic cultures were initiated from leaves of in vitro grown shoot cultures and used as target tissues for Agrobacterium-mediated genetic transformation. A green fluorescent protein/neomycin phosphotransferase II (gfp/nptII) fusion gene that allowed for simultaneous selection of transgenic cells based on GFP fluorescence and kanamycin resistance was used to optimize parameters influencing genetic transformation. It was determined that both proembryonal masses (PEM) and mid-cotyledonary stage somatic embryos (SE) were suitable target tissues for co-cultivation with Agrobacterium as evidenced by transient GFP expression. Kanamycin at 100 mg l⁻¹ in the culture medium was effective in suppression of non-transformed tissue and permitting the growth and development of transgenic cells, compared to 50 or 75 mg l⁻¹, which permitted the proliferation of more non-transformed cells. Transgenic plants of “Alachua” and “Carlos” were recovered after secondary somatic embryogenesis from primary SE explants co-cultivated with Agrobacterium. The presence and stable integration of transgenes in transgenic plants was confirmed by PCR and Southern blot hybridization. Transgenic plants exhibited uniform GFP expression in cells of all plant tissues and organs including leaves, stems, roots, inflorescences and the embryo and endosperm of developing berries.     

Regulated Expression of a Cytokinin Biosynthesis Gene IPT Delays Leaf Senescence and Improves Yield under Rainfed and Irrigated Conditions in Canola (Brassica napus L.)

Delay of leaf senescence through genetic modification can potentially improve crop yield, through maintenance of photosynthetically active leaves for a longer period. Plant growth hormones such as cytokinin regulate and delay leaf senescence. Here, the structural gene (IPT) encoding the cytokinin biosynthetic enzyme isopentenyltransferase was fused to a functionally active fragment of the AtMYB32 promoter and was transformed into canola plants. Expression of the AtMYB32xs::IPT gene cassette delayed the leaf senescence in transgenic plants grown under controlled environment conditions and field experiments conducted for a single season at two geographic locations. The transgenic canola plants retained higher chlorophyll levels for an extended period and produced significantly higher seed yield with similar growth and phenology compared to wild type and null control plants under rainfed and irrigated treatments. The yield increase in transgenic plants was in the range of 16% to 23% and 7% to 16% under rainfed and irrigated conditions, respectively, compared to control plants. Most of the seed quality parameters in transgenic plants were similar, and with elevated oleic acid content in all transgenic lines and higher oil content and lower glucosinolate content in one specific transgenic line as compared to control plants. The results suggest that by delaying leaf senescence using the AtMYB32xs::IPT technology, productivity in crop plants can be improved under water stress and well-watered conditions.     

Over-expression of the Arabidopsis Na+/H+ antiporter gene in Populus deltoides CL?×?P. euramericana CL “NL895” enhances its salt tolerance

Soil salinity is a serious problem worldwide. It is necessary to improve the salt tolerance of plants to avoid the progressive deterioration of saline soil. We showed that the over-expression of AtNHX1 improves salt tolerance in a transgenic poplar (Populus deltoides CL × P. euramericana CL “NL895”) under mannose selection. Four transgenic poplar plants were obtained. Southern blot analysis showed that the pmi gene had integrated into the genome of the poplar. RT-PCR confirmed that AtNHX1 could be expressed normally in the transgenic plants. When tested for salt tolerance by NaCl stress, we measured a 100% increase in Na⁺ content in the three transgenic lines (T18, T50, T98) significantly higher than the 33% increase seen in wild-type plants. The chlorophyll content of the transgenic plants was not altered significantly, while the chlorophyll content in the control plants showed a small decrease. MDA content was decreased in the transgenic plants. These results show that the AtNHX1 gene may enhance salt tolerance due to increased vacuolar compartmentalization of sodium ions.