Repression of a novel isoform of disproportionating enzyme (stDPE2) in potato leads to inhibition of starch degradation in leaves but not tubers stored at low temperature

A potato (Solanum tuberosum) cDNA encoding an isoform of disproportionating enzyme (stDPE2) was identified in a functional screen in Escherichia coli. The stDPE2 protein was demonstrated to be present in chloroplasts and to accumulate at times of active starch degradation in potato leaves and tubers. Transgenic potato plants were made in which its presence was almost completely eliminated. It could be demonstrated that starch degradation was repressed in leaves of the transgenic plants but that cold-induced sweetening was not affected in tubers stored at 4 degrees C. No evidence could be found for an effect of repression of stDPE2 on starch synthesis. The malto-oligosaccharide content of leaves from the transgenic plants was assessed. It was found that the amounts of malto-oligosaccharides increased in all plants during the dark period and that the transgenic lines accumulated up to 10-fold more than the control. Separation of these malto-oligosaccharides by high-performance anion-exchange chromatography with pulsed-amperometric detection showed that the only one that accumulated in the transgenic plants in comparison with the control was maltose. stDPE2 was purified to apparent homogeneity from potato tuber extracts and could be demonstrated to transfer glucose from maltose to oyster glycogen.     

Co-expression of Pennisetum glaucum vacuolar Na⁺/H⁺ antiporter and Arabidopsis H⁺-pyrophosphatase enhances salt tolerance in transgenic tomato

Salinity is one of the major abiotic stresses affecting plant productivity. Tomato (Solanum lycopersicum L.), an important and widespread crop in the world, is sensitive to moderate levels of salt in the soil. To generate tomato plants that can adapt to saline soil, AVP1, a vacuolar H(+)-pyrophosphatase gene from Arabidopsis thaliana, and PgNHX1, a vacuolar Na(+)/H(+) antiporter gene from Pennisetum glaucum, were co-expressed by Agrobacterium tumefaciens-mediated transformation. A sample of transformants was self-pollinated, and progeny were evaluated for salt tolerance in vitro and in vivo. It is reported here that co-expression of AVP1 and PgNHX1 confers enhanced salt tolerance to the transformed tomato compared with the AVP1 and PgNHX1 single gene transgenic plants and the wild-type. These transgenic plants grew well in the presence of 200 mM NaCl while wild-type plants exhibited chlorosis and died within 3 weeks. The transgenic line co-expressing AVP1 and PgNHX1 retained more chlorophyll and accumulated 1.4 times more proline as a response to stress than single gene transformants. Moreover, these transgenic plants accumulated a 1.5 times higher Na(+) content in their leaf tissue than the single gene transformants. The toxic effect of Na(+) accumulation in the cytosol is reduced by its sequestration into the vacuole. The physiological analysis of the transgenic lines clearly demonstrates that co-expression of AVP1 and PgNHX1 improved the osmoregulatory capacity of double transgenic lines by enhanced sequestration of ions into the vacuole by increasing the availability of protons and thus alleviating the toxic effect of Na(+).     

Overexpression of NHX1s in transgenic Arabidopsis enhances photoprotection capacity in high salinity and drought conditions

In this study, we observed that transgenic plants overexpressing NHX1 from different organisms grew well in the presence of 200 mM NaCl and also under water deprivation, while the wild-type plants exhibited chlorosis and growth inhibition. The photosynthesis activity of five kinds of transgenic plants was higher than the wild-type plants. The leaf water potential was less negative for wild-type than for transgenic plants. Moreover, these transgenic plants accumulated more Na⁺ and K⁺ in their leaf tissue than the wild-type plants. The toxic effects of Na⁺ accumulation in the cytosol are reduced by its sequestration into the vacuole. In addition, the thermal dissipation and ROS scavenging components increased in all transgenic Arabidopsis plants compared with that in non-transgenics. The salt tolerance of transgenic plants was passed on to the offsprings to the T₅ generation. Furthermore, it should be noted that in transgenic Arabidopsis plants, overexpression of NHX1s from dicots showed higher salt and drought tolerance than that from wheat.     

Enhanced arsenic tolerance of transgenic eastern cottonwood plants expressing gamma-glutamylcysteine synthetase

Arsenic is a metalloid that occurs naturally at parts per million (ppm) levels in the earth's crust. Natural and human activities have contributed to arsenic mobilization and increased concentration in the environment, such that World Health Organization guidelines for arsenic levels in drinking water are exceeded at many locations, worldwide. This translates into an increased risk of arsenic-related illnesses for millions of people. Recent studies demonstrate that increasing thiol-sinks in transgenic plants by overexpressing the bacterial gamma-glutamylcysteine synthetase (ECS) gene results in a higher tolerance and accumulation of metals and metalloids such as cadmium, mercury, and arsenic. We used Agrobacterium-mediated transformation to genetically engineer eastern cottonwood with a bacterial ECS gene. Eastern cottonwood plants expressing ECS had elevated thiol group levels, consistent with increased ECS activity. In addition, these ECS-expressing plants had enhanced growth on levels of arsenate toxic to control plants in vitro. Furthermore, roots of ECS-expressing plants accumulated significantly more arsenic than control roots (approximately twice as much), while shoots accumulated significantly less arsenic than control shoots (approximately two-thirds as much). We discuss potential mechanisms for shifting the balance of plant arsenic distribution from root accumulation to shoot accumulation, as it pertains to arsenic phytoremediation.     

Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants

Gene constructs were designed to test the effect of the endoplasmic reticulum (ER)-targeting signal, KDEL, on the level of accumulation of a foreign protein in transgenic plants. The gene for the pea seed protein vicilin was modified by the addition of a sequence coding for this tetrapeptide at its carboxyl terminus. The altered gene was placed under the control of a CaMV 35S promoter and its expression in the leaves of both tobacco and lucerne (alfalfa) was compared with that of an equivalent vicilin construct lacking the KDEL-coding sequence. The presence of the ER-targeting signal led to a greatly enhanced accumulation of the heterologous protein. In lucerne and tobacco leaves, the level of vicilin-KDEL protein was 20 and 100 times greater than that of the unmodified vicilin, respectively. These differences in expression level could not be explained by corresponding differences in the steady-state levels or the translatability of the mRNAs. However, when the stability of vicilin and vicilin-KDEL proteins was compared in their respective transgenic hosts, unmodified vicilin was found to be degraded with a half-life of 4.5 h while vicilin-KDEL was much more stable with a half-life of more than 48 h. Immunogold labelling of leaf tissues from transgenic lucerne and tobacco showed the presence of vicilin associated with large aggregates within the ER lumen of vicilin-KDEL plants. No such aggregates were detected in transgenic plants expressing wild-type vicilin. It is concluded that the carboxy-terminal KDEL caused the retention of the modified vicilin in the ER, and that this retention led to the increased stability and higher level of accumulation of vicilin-KDEL in leaves of transgenic plants.     

Nickel accumulation in rape shoots (Brassica napus L.) increased by putrescine

Application of exogenous putrescine (Put) increases nickel accumulation in rape shoots (Brassica napus), improving potential for phytoremediation of contaminated soils. Plants were grown within a growth chamber in water culture for five weeks, then 250-500 microM NiCl2 was added to rooting media. Within 5 days of treatment, damaging effects of nickel manifested as a reduction of root system size and a decrease of Cu and especially Fe content in young leaves. Spraying leaves of adult plants with Put markedly reduced toxic effects of Ni on root growth, enhanced leaf supply with Fe, and increased Ni content in young leaves by 2.5 times. Plant growth in medium with elevated levels of Ni stimulated accumulation of endogenous spermidine (Spd), spermine (Spm), and especially Put (by 4 times as compared with the control). Results suggest that Ni-induced accumulation of endogenous polyamines in rape leaves is caused by activation of long-distance metal transport within the plant and reduction of its toxicity due to Put chelating action.     

Effects of free proline accumulation in petunias under drought stress

Petunias (Petunia hybrida cv. 'Mitchell') accumulate free proline (Pro) under drought-stress conditions. It is therefore believed that Pro acts as an osmoprotectant in plants subjected to drought conditions. Petunia plants were transformed by Delta(1)-pyrroline-5-carboxylate synthetase genes (AtP5CS from Arabidopsis thaliana L. or OsP5CS from Oryza sativa L.). The transgenic plants accumulated Pro and their drought tolerance was tested. The Pro content amounted to 0.57-1.01% of the total amino acids in the transgenic plants, or 1.5-2.6 times that in wild-type plants grown under normal conditions. The transgenic plant lines tolerated 14 d of drought stress, which confirms that both P5CS transgenes had full functionality. Exogenous L-Pro treatment caused the plants to accumulate Pro; plants treated with 5 mM L-Pro accumulated up to 18 times more free Pro than untreated plants. Exogenous L-Pro restricted the growth of wild-type petunias more than that of Arabidopsis plants. The capacity for free Pro accumulation might depend on the plant species. The growth of petunia plants was influenced not only by the Pro concentration in the plants, but by the ratio of the Pro content to the total amino acids, because the growth of the transgenic petunia plants appeared normal.     

Expression of a fungal phytase gene in Nicotiana tabacum improves phosphorus nutrition of plants grown in amended soils

Transgenic Nicotiana tabacum plants expressing a chimeric phytase gene (ex::phyA) from the soil fungus Aspergillus niger were generated. Three independently transformed lines showed increased extracellular phytase activity compared with a vector control and wild-type plants, both of which had no detectable extracellular phytase. Transgenic N. tabacum plants grown in sterile agar supplied with phosphorus (P) as phytate accumulated 3.7-fold more P than vector control plants. Despite this, the expression of ex::phyA in plants did not lead to an improved accumulation of P from two unamended P-deficient soils. However, when soils were amended with either phytate or phosphate and lime, transgenic plants accumulated up to 52% more P than controls. Positive responses by transgenic plants were, in some instances, coincident with a putative increase in soil phytate. We conclude that the development of plants that exude phytase to the soil may not ensure improved plant P nutrition, as the availability of phytate in the soil also appears to be critical. Nevertheless, if plants that express ex::phyA are combined with soil amendments that promote the availability of phytate, there is the potential to enhance the P nutrition of crop plants and to improve the efficiency of P fertilizer use in agricultural systems.     

Overexpression of an H+-PPase gene from Thellungiella halophila in cotton enhances salt tolerance and improves growth and photosynthetic performance

Salinity is one of the major environmental factors limiting plant growth and productivity. An H(+)-PPase gene, TsVP from Thellungiella halophila, was transferred into cotton (Gossypium hirsutum) in sense and antisense orientations under control of the cauliflower mosaic virus (CaMV) 35S promoter. Southern and Northern blotting analysis showed that the sense or antisense TsVP were integrated into the cotton genome and expressed. Transgenic plants overexpressing the vacuolar H(+)-PPase were much more resistant to 150 and 250 mM NaCl than the isogenic wild-type plants. In contrast, the plants from the antisense line (L-2), with lower H(+)-PPase activity, were more sensitive to salinity than the wild-type plants. Overexpressing TsVP in cotton improved shoot and root growth and photosynthetic performance. These transgenic plants accumulated more Na(+), K(+), Ca(2+), Cl(-) and soluble sugars in their root and leaf tissues under salinity conditions compared with the wild-type plants. The lower membrane ion leakage and malondialdehyde (MDA) level in these transgenic plants suggest that overexpression of H(+)-PPase causes the accumulation of Na(+) and Cl(-) in vacuoles instead of in the cytoplasm, thus reducing their toxic effects. On the other hand, the increased accumulation of ions and sugars decreases the solute potential in cells, and facilitates water uptake under salinity, which is an important mechanism for the increased salt tolerance in TsVP-overexpressing cotton.     

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.     

Overexpression of a new rice vacuolar antiporter regulating protein OsARP improves salt tolerance in tobacco

We examined the function of the rice (Oryza sativa L.) antiporter-regulating protein OsARP by overexpressing it in tobacco (Nicotiana tabacum L.). In public databases, this protein was annotated as a putative Os02g0465900 protein of rice. The OsARP gene was introduced into tobacco under the control of the cauliflower mosaic virus 35S promoter. The transformants were selected for their ability to grow on medium containing kanamycin. Incorporation of the transgene in the genome of tobacco was confirmed by PCR, and its expression was confirmed by Western blot analysis. Transgenic plants had better growth and vigor than non-transgenic plants under salt stress in vitro. Overexpression of OsARP in transgenic tobacco plants resulted in salt tolerance, and the plants had a higher rate of photosynthesis and effective PSII photon yield when compared with the wild type. The OsARP protein was localized in the tonoplast of rice plants. Transgenic plants accumulated more Na+ in their leaf tissue than did wild-type plants. It is conceivable that the toxic effect of Na+ in the cytosol might be reduced by sequestration into vacuoles. The rate of water loss was higher in the wild type than in transgenic plants under salt stress. Increased vacuolar solute accumulation and water retention could confer salt tolerance in transgenic plants. Tonoplast vesicles isolated from OsARP transgenic plants showed Na+/H+ exchange rates 3-fold higher than those of wild-type plants. These results suggest that OsARP on the tonoplasts plays an important role in compartmentation of Na+ into vacuoles. We suggest that OsARP is a new type of protein participating in Na+ uptake in vacuoles.     

Evaluation of Transgenic Poplars Over-Expressing Enzymes of Glutathione Synthesis for Phytoremediation of Cadmium

Abstract: Recently, phytoremediation of soils polluted with heavy metals has received a lot of attention. Since glutathione (GSH) and its derivatives (e.g., phytochelatins) play a major role in plant defence against environmental pollutants, we tested the effects of over-expression of bacterial genes for GSH synthesis in poplar on cadmium accumulation. A pilot experiment with CdCl₂ in hydroponics revealed that poplars over-expressing γ-glutamylcysteine synthetase (γ-ECS) accumulated significantly more Cd in root tissue than wild type or glutathione synthetase over-expressing poplars. To test the partitioning of Cd in different organs, poplar lines over-expressing γ-ECS in the cytosol and in chloroplasts were treated with 0.2 mM CdCl₂ in hydroponics. Significant amounts of Cd were translocated to leaves, but significant differences in Cd accumulation were not observed between transgenic and wild type plants. To evaluate these lines for large-scale phytoremediation of cadmium, plants were treated with 2 mM Cd in soil. Over a four-week period, the poplar plants were able to accumulate up to 5.3 mg Cd. Most remarkably, in young leaves of both transgenic lines, Cd was accumulated to concentrations 2.5 - 3 times higher than in the wild type. The increased allocation of cadmium to the young leaves represents a potentional advantage for the phytoremediation process using the same plants over several vegetation periods. The use of transgenic poplar lines with enhanced glutathione production capacity seems to be of particular advantage in highly polluted soils.     

The absorption, translocation and distribution of the herbicide glyphosate in maize expressing the CP-4 transgene

Maize (Zea mays L. var. Bonnie) transformed with a gene encoding a 5-enolpyruvylshikimate 3-phosphate synthase with altered sensitivity showed over 100-fold greater resistance to the herbicide glyphosate (N-[phosphonomethyl]glycine) in comparison with its non-transformed progenitor (parental control) at the third-leaf stage. Studies with [¹⁴C]-glyphosate at a dosage lethal to the parental control, but sublethal to the transgenic, revealed that a maximum of 45-65% of the applied dose was absorbed, with greater absorption occurring in transgenic plants. Translocation of glyphosate was closely related to its absorption (r value 0.956) with approximately 15% more of the applied dose being mobilized in transgenic plants than the parental controls. Analysis of electronic autoradiograms along the treated leaf lamina found discrete internal regions of glyphosate accumulation closely associated with the site of application. These regions contained lower amounts of glyphosate present in the treated leaf lamina was almost completely translocated in transgenic plants, while in the parental controls more remained and the leaf became necrotic. In both types of maize there was a small accumulation of herbicide in the tip region of the leaf which was not mobilized. Younger shoot tissues and roots were major sinks for translocated glyphosate accumulating approximately 25-40% of the applied dose depending upon treatment. In the parental control, equal amounts of glyphosate were found distributed between young shoot tissues and roots; while in transgenic plants, the young shoot tissue accumulated around three times more glyphosate than the roots. In both plant types, glyphosate was localized in the meristems and young, actively growing leaves. Specific glyphosate activity (the amount of glyphosate per unit dry weight of tissue) in the major sinks of the transgenic declined towards the end of the treatment period but remained relatively constant in the parental control. In conclusion, enhancing glyphosate resistance by genetic transformation influenced the absorption, translocation and distribution of this herbicide in whole plants.Keywords: Zea mays, glyphosate (N-[phosphonomethyl]-glycine), transgenic, absorption, translocation, source-sink.      

Transgenic Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) plants expressing an <Emphasis Type="Italic">Arabidopsis</Emphasis> phytochelatin synthase (<Emphasis Type="Italic">AtPCS1</Emphasis>) exhibit enhanced As and Cd tolerance

Phytochelatins (PCs) are post-translationally synthesized thiol reactive peptides that play important roles in detoxification of heavy metal and metalloids in plants and other living organisms. The overall goal of this study is to develop transgenic plants with increased tolerance for and accumulation of heavy metals and metalloids from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A FLAG-tagged AtPCS1 gDNA, under its native promoter, is expressed in Indian mustard, and transgenic pcs lines have been compared with wild-type plants for tolerance to and accumulation of cadmium (Cd) and arsenic (As). Compared to wild type plants, transgenic plants exhibit significantly higher tolerance to Cd and As. Shoots of Cd-treated pcs plants have significantly higher concentrations of PCs and thiols than those of wild-type plants. Shoots of wild-type plants accumulated significantly more Cd than those of transgenic plants, while accumulation of As in transgenic plants was similar to that in wild type plants. Although phytochelatin synthase improves the ability of Indian mustard to tolerate higher levels of the heavy metal Cd and the metalloid As, it does not increase the accumulation potential of these metals in the above ground tissues of Indian mustard plants.     

Overexpression of selenocysteine methyltransferase in Arabidopsis and Indian mustard increases selenium tolerance and accumulation

A major goal of phytoremediation is to transform fast-growing plants with genes from plant species that hyperaccumulate toxic trace elements. We overexpressed the gene encoding selenocysteine methyltransferase (SMT) from the selenium (Se) hyperaccumulator Astragalus bisulcatus in Arabidopsis and Indian mustard (Brassica juncea). SMT detoxifies selenocysteine by methylating it to methylselenocysteine, a nonprotein amino acid, thereby diminishing the toxic misincorporation of Se into protein. Our Indian mustard transgenic plants accumulated more Se in the form of methylselenocysteine than the wild type. SMT transgenic seedlings tolerated Se, particularly selenite, significantly better than the wild type, producing 3- to 7-fold greater biomass and 3-fold longer root lengths. Moreover, SMT plants had significantly increased Se accumulation and volatilization. This is the first study, to our knowledge, in which a fast-growing plant was genetically engineered to overexpress a gene from a hyperaccumulator in order to increase phytoremediation potential.     

Enhancement of NADP-malic enzyme in transgenic rice induced the accumulation of reactive oxygen species

It had been demonstrated that the photosynthetic photodamage, such as photoinhibition and photooxidation, was enhanced in transgenic rice plants overexpressing NADP-malic enzyme (ME). However, its physiological base has not been investigated. In order to elucidate the physiological elements contributed to the enhancement of photodamage in NADP-ME transgenic rice plants, some physiological indices related to reactive oxygen species (ROS) accumulation were studied using the T₁ progeny of transgenic rice plants. The results showed that more ROS such as O ₂ ^−. and H₂O₂ were accumulated in transgenic rice plants, which enhanced photooxidation, while the accumulation of malondialdehyde in transgenic rice plants was not evident as compared with the wild-type plants. The measurement of NADPH/NADP ratios in leaves showed that transgenic rice plants had a higher ratio than untransformed rice plants. Based on these data, we speculated that overexpression of NADP-ME led to the deficiency of NADP and overreduction of photosystem I, which induced the accumulation of ROS in the transgenic rice plants, and just ROS were accounted for plant sensitivity to photooxidation. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 364–370. The text was submitted by the author in English.     

Expression of E. coli inorganic pyrophosphatase in transgenic plants alters photoassimilate partitioning

Transgenic plants were constructed expressing a novel cytosolic inorganic pyrophosphatase in order to reduce the cytosolic pyrophosphate content. To this end the Escherichia coli gene ppa encoding inorganic pyrophosphatase was cloned between the 35S CaMV promoter and the poly(A) site of the octopine synthase gene and transferred into tobacco and potato plants by Agrobacterium-mediated gene transfer. Regenerated plants were tested for the expression of the ppa gene by Northern blots and activity gels. Plants expressing active inorganic pyrophosphatase showed a dramatic change in photoassimilate partitioning. In both transgenic tobacco and potato plants the ratio between soluble sugars and starch was increased by about 3-4-fold in source leaves as compared with the wild-type. However, whereas source leaves of transgenic tobacco plants accumulated much higher levels of glucose (up to 68-fold), fructose (up to 24-fold), sucrose (up to 12-fold) and starch (up to 8-fold) this was not observed in potato plants where the change in assimilate partitioning in source leaves was due to an increase of about 2-fold in sucrose and a reduction in starch content. Expression of the cytosolic inorganic pyrophosphatase in tobacco results in stunted growth of vegetatively growing plants due to a reduced internode distance. Upon flowering the transgenic plants increase their growth rate, reaching almost the same height as control plants at the end of the growth period. Old source leaves accumulate up to 100-fold more soluble sugars than control leaves. This increase in soluble sugars is accompanied by a reduction in chlorophyll content (up to 85%). Transgenic potato plants showed a less dramatic change in their growth behaviour. Plants were slightly reduced in size, with stems more highly branched. Tuber number increased 2-3-fold, but tuber weight was lower resulting in no net increase in fresh weight.