Since nitrogen (N) is the most essential nutrient for plants and a major limiting factor in plant productivity, doubling agricultural food production worldwide over the past four decades is associated with a 20-fold increase in N fertilizer use. As a consequence, use of N fertilizers in agriculture has already shown a number of detrimental environmental impacts. Therefore, the need to reduce N fertilizer pollution is strengthening the importance of improving the nitrogen use efficiency (NUE) of crop plants. The development of crop plants that take-up and assimilate N more efficiently would reduce the need for N fertilizers and positively influence the environment. Here, we discuss recent developments in the genetic manipulations of NUE in crop plants.

Background
Crop plants, especially grown for protein content and grain yield, require large quantities of inorganic N fertilizers¹. Consequently, in the last 50 years the N fertilization of crop plants worldwide has increased more than 20-fold. However, use of this fertilizer is generally inefficient, as only about a third of the fertilizer applied is actually absorbed by crops, and 50 – 70% is lost from the plant-soil system². Unused fertilizer can leach into the environment, where it induces algal blooms, contaminates drinking water, and depletes aquatic oxygen to create dead zones, like those found in the Gulf of Mexico. Recently, Johnson and colleagues³ showed that elevated nutrient inputs into aquatic ecosystems due to heavy use of N and phosphorus leads to eutrophication and increases pathogenic infection in amphibians. Because of the heavy use of N fertilizers, which is one of the major costs associated with the production of high-yielding crops and is the source of environmental damage due to excess N that is not taken up by plants^4,5, there is significant interest in genetic engineering crops to improve NUE^6-8.

Engineering plants with transport gene systems
Crop plants obtain N from the soil primarily as nitrate or ammonia, although some plants utilize amino acids as significant sources of N. Following uptake by specific transporters located in the root cell membrane, nitrate is reduced to ammonium through the combined action of nitrate reductase (NR) and nitrite reductase (NiR). In higher plants, the expression of the NR genes is influenced by several external and endogenous factors and is highly regulated at the transcriptional as well as post-translational levels⁹. The overexpression of either the NR or the NiR gene in plants increases mRNA levels and often affects N uptake. However, the increased uptake of N does not seem to increase the yield or growth of plants, regardless of the N source^6,7. This is believed to be due, in part, to the complex regulation of both NR and the pathway as a whole. Recently, Lea et al.¹⁰ demonstrated that post-translational regulation of NR strongly affects the levels of free amino acids, ammonium, and nitrate, whereas transcriptional regulation has only minor influence. Plants expressing fully unregulated NR accumulate high concentrations of asparagine and glutamine in leaves; however these transgenic plants grow and developed normally, despite having an NR enzyme that is active during both light and dark periods.

Glutamine synthetase and glutamate synthase gene systems
In higher plants, glutamine synthetase (GS) is represented by two groups of proteins—the cytosolic and plastidic forms¹¹. A large number of studies on various plant species including both monocots and dicots show that cytosolic GS (GS1) is encoded by a complex multigene family, whereas plastidic GS (GS2) is encoded by a single gene¹. Glutamate synthase (GOGAT) occurs as two distinct isoforms—ferridoxin and NADH-dependent—both of which are located in the plastid.

Since the discovery of the role of GS/GOGAT in ammonium assimilation in higher plants¹², there has been great interest in understanding the mechanisms controlling the regulation of this pathway¹³. Mutants or transgenic plants with altered levels of GS/GOGAT are used to determine the effects of these proteins on plant development and to study the expression of the different members of the GS multigene family¹⁴.

Although several studies demonstrate that an increase in GS activity in transgenic plants has no effect on the phenotype, other researchers show a direct correlation between an enhanced GS activity in transgenic plants and an increase in biomass or yield, upon incorporating a novel gs1 construct^6,8,15. For example, tobacco plants overexpressing the gs1 gene demonstrate increased fresh weight, dry weight, and leaf protein that is directly correlated with an increased level of GS in leaves¹⁶. Fei et al.¹⁷ produced transgenic peas overexpressing the cytosolic gs1 gene and demonstrated that these transgenic lines have a two- to eightfold increase in GS activity in roots. Transgenic pea plants overexpressing the gs15 gene under the control of a root specific promoter also demonstrate an increased biomass and N content¹⁸. However, inconsistent growth effects in the transgenic plants are also observed. Recently, poplar trees transformed with a conifer gs1a gene demonstrate significant increases in leaf area, dry weight, and plant height, both in controlled environmental and field conditions. Interestingly, the differences are more striking at a low nitrate concentration. In addition, higher rates of ¹⁵N incorporation into the transgenic plants further demonstrate that the transformed plants have increased NUE¹⁹.

In comparison to GS, few reports have described the production of transgenic plants overexpressing GOGAT genes. The most interesting results were obtained by Yamaya et al.²⁰ who overexpressed OsNADH-GOGAT1 in rice under the control of its own promoter and found that transgenic rice plants show an increase in spikelet weight (up to 80%). Plant heights and spikelet number are unaffected. This study shows that overexpression of NADH-GOGAT1 can be used as a key step for N use and grain filling in rice and other cereal crops.

Engineering plants with other gene systems regulating N metabolism
Over the past few years, attention was focused on the enzyme asparagine synthetase (AS), which catalyzes the formation of asparagine (Asn) and glutamate from glutamine (Gln) and aspartate. In higher plants, AS is encoded by a small gene family²¹. Together with GS, AS is believed to play a crucial role in primary N metabolism^13,22. The observation that the levels of AS transcripts and polypeptides in the transgenic nodules of Medicago truncatula increase when GS is reduced suggests that AS can compensate for the reduced GS ammonium assimilatory activity²². However, the same authors also demonstrated that GS activity is essential for maintaining the higher level of AS. Thus, GS is required to synthesize enough Gln to support Asp biosynthesis via NADH-GOGAT and AspAT²².

A reduction in GS activity in transgenic Lotus japonicus is also correlated with an increase in asparagine content²³, supporting the hypothesis that when GS becomes limiting, AS may be important in controlling the flux of reduced N into plants. With the aim of increasing Asn production in plants and to study the role of AS, several researchers attempted to clone AS genes and to examine the corresponding gene expression in plants. For example, Lam and colleagues²⁴ overexpressed the ASN1 gene in Arabidopsis and demonstrated that the transgenic plants have enhanced soluble seed protein content, enhanced total protein content, and better growth on N-limiting medium. Arabidopsis plants overexpressing the ASN2 gene accumulate less endogenous ammonium than wild-type plants when grown on medium containing 50-mM ammonium. When plants are subjected to high light irradiance, ammonium levels increase²⁵. Transgenic Arabidopsis plants overexpressing the maize Dof1 transcription factor demonstrate not only better growth under N limiting conditions, but also enhanced N assimilation²⁶. This study indicates that signaling processes may provide an attractive route for metabolic engineering. In comparison to GS/GOGAT enzymes, the physiological role of glutamate dehydrogenase (GDH) has been less clear²⁷. In an attempt to investigate the role of GDH by expressing a bacterial gdhA gene from E. coli in tobacco, Ameziane et al.²⁸ found that biomass production is consistently increased in gdhA transgenics, regardless of whether they are grown under controlled conditions or in the field.

The challenge of manipulating N remobilization
Remobilization of N in plants is a very complex metabolic process and is of major importance for plant productivity because it recycles organic N to young developing leaves and storage organs²⁹. Therefore, in cereals and other crops, grain yield is based not only on nitrate uptake before flowering but also on the remobilization of leaf N during seed maturation. In rice, approximately 80% of the total N in the panicle arises from remobilization through the phloem from senescing organs³⁰. During the past few years, efforts have been made to identify genes encoding proteins that are specifically activated during the remobilization of N, carbon, and minerals during leaf senescence³¹. In addition, several laboratories are studying the biochemical mechanisms involved in N export and import from source and sink leaves during senescence^29,32.

Since cytosolic GS (GS1) is only induced during leaf senescence, it has therefore been suggested that this enzyme reassimilates ammonium released from protein hydrolysis³³. Several studies using transgenic tobacco demonstrate that genetic manipulation influences plant phenotype and amino acid metabolism when N is limiting³⁴. During N remobilization in cereals, GS1 facilitates the synthesis of Gln, which is the major form of reduced N in phloem sap, and NADH-GOGAT1 is important in developing sink organs for the remobilization of Gln in rice⁷. Thus, the synthesis of Gln in senescing organs is considered a key step in N recycling.

A large increase in the amino acid content of roots (primarily) and shoots and premature flowering are observed in Lotus corniculatus overexpressing a soybean gene (gs15), which encodes cytosolic GS³⁵. ¹⁵N labeling experiments further demonstrate that both ammonium uptake in roots and the subsequent translocation of amino acids to shoots is lower in plants overexpressing gs15. These results suggest that the accretion of ammonium and amino acids in roots is due to shoot protein degradation. These results further confirm that N remobilization is induced artificially by the overexpression of gs15.

When transgenic wheat lines expressing the Phaseolus vulgaris gs1 gene are grown in pots to maturity and their productivity analyzed, they demonstrate an enhanced capacity to accumulate N in the plant. Measurement of the total N content of tissue at harvest shows that transgenic plants with extra GS1 protein accumulate more N in their shoots and grain³⁶. Although only one transgenic line showed improved N assimilation in one study, this indicates that genetic transformation of plants with GS may have a practical effect on NUE.

Recently, the roles of two genes encoding cytosolic maize GS1 (gln1-3 and gln1-4) were investigated in detail by examining the impact of knockout mutations on kernel yield and by overexpressing gln1-3 in maize³⁷. The authors found that gln1-4 gln1-3 double mutants display reduced kernel size and reduced kernel number, with no reduction in shoot biomass production at maturity. When maize is genetically transformed by constitutively overexpressing gln1-3 using a cassava vein mosaic virus promoter, a significant increase in grain yield is observed (~30%). Again, there are no significant differences in shoot dry matter production between WT plants and the transgenic lines, which suggests the specific impact of gln1-3 on grain production. Transgenic maize plants overexpressing the gln1-3 gene produce greater kernel numbers under both high and low N conditions when compared to wild type plants¹⁵. These studies on maize clearly suggest that GS1 plays an important role in kernel yield under high and low N fertilization. The reaction catalyzed by GS1, therefore, may be one of the key elements controlling crop yield.

In rice, GS1 knock-out mutants made by inserting the retrotransposon Tos17 into exon-8 or exon-10 of Osgs1;1 exhibit a severe reduction in growth and grain filling when grown using normal N fertilizer concentration. Reintroduction of the Osgs1;1 cDNA under the control of its own promoter into the mutants successfully complements the slow growth phenotype. This study further indicates that GS1;1 is important for normal growth and grain filling in rice. GS1;2 and GS1;3 are not able to compensate for the function of GS1;1^38,30.

Summary
Studies with transgenic plants overexpressing genes affecting the N metabolism pathway suggest it is possible to improve or manipulate N metabolism and the growth phenotype of plants, which can improve the NUE of crop plants. However, in spite of studies conducted over the past few years both at the whole plant level and using transgenic plants, understanding the mechanisms involved in N remobilization during leaf senescence and remobilization is still at a preliminary stage and requires more research.

In their excellent review article, Hirel and Lemaire³⁴ emphasize that for relatively long periods during vegetative growth, plant nutrition is near a steady state condition. However, after anthesis, crops experience a rapid exhaustion of the available N in soil and therefore grain filling has to be directly supported by N recycling. An improved understanding of the transition between N assimilation and N recycling will undoubtedly be of tremendous importance in applying transgenic approaches to improving the NUE of crop plants.

In order to further identify and understand the regulation of the genes involved in enhancing NUE, proper evaluation of the combined genetic and transgenic approaches to improving NUE should be required as a component of any crop improvement program. The benefits of growing NUE-efficient crops will not be realized until breeders evaluate N metabolism and nitrogen use efficiency in economically important crop plants. Given that the global human population is expected to reach ten billion by 2070, feeding everyone will require the more efficient use of agricultural lands, and creating crops with enhanced nutrient uptake will be one component in achieving this goal.

EXPRESSING HEPATITIS B VIRUS SURFACE ANTIGEN IN RICE
Janaki Krishna

Hepatitis B virus, which infects the liver of hominidae, including humans, is one of many unrelated viruses that cause viral hepatitis. The proportion of the world’s population currently infected with the virus is estimated at 3% to 6%. Chronic hepatitis B infection may eventually cause liver cirrhosis and liver cancer, a fatal disease with a very poor response to current chemotherapy. The disease was originally known as ‘Serum Hepatitis’ and has caused epidemics in parts of Asia and Africa, and is endemic in China and various other parts of Asia. The infection is preventable by vaccination, and after the development of a commercial recombinant vaccine derived from yeast, there was a decline in the spread of HBV infection. Because plants may be promising bioreactors for producing vaccines, much research is geared toward the cost effective production of recombinant surface antigens in plants. For example, researchers from Fudan University, Jiao Tong University, and the Institute for Biological Sciences, Shanghai, China recently reported producing a novel hepatitis B vaccine in rice seeds.

The team constructed a rice endosperm-specific vector containing the SSI gene, which expresses a modified hepatitis B virus (HBV) surface antigen (HBsAg). To do this, a 2.8 kb SS1 expression cassette was cloned into p^CAMBIA1300 to produce the p1300GSS1 plant binary vector. Transgenic rice plants (Oryza sativa L.) were produced through Agrobacterium mediated transformation. Incorporation of the genome into the target gene was confirmed by PCR and Southern blot analysis. Also, RNA dot blot analyses were performed to further test whether the fused SS1 gene was specifically expressed in rice seeds. The amount of recombinant SS1 protein in transgenic rice seeds was measured by quantitative ELISA. A CsCl gradient analysis was performed to find out whether the introduced recombinant SS1 in rice plants formed virus-like particle (VLP) structures. To observe VLPs in the recombinant SS1 protein fraction, solid phase immune electron microscopy was used.

Finally, the immunogenic response of the recombinant SS1 protein was tested in adult BALB/c female mice. In this experiment, mice aged 6 to 8 weeks were immunized intraperitonially three times at two-week intervals with the freeze-dried total protein of all the transgenic rice seeds. Each dose contained 0.5 µg of recombinant SS1 protein emulsified with 200 µl of complete Freund’s adjuvant in a final volume of 400 µl at the first immunization, and with incomplete Freund’s adjuvant at subsequent immunizations. A control experiment was also carried out with another group of eight adult mice, immunizing them with the total protein of non-transgenic rice seeds. Because the aim of the research was to generate recombinant protein with S (HBV surface protein) and preS1 (presurface 1 region) epitope immunogenicity, the presence of antibodies against S and preS1 in mice sera was tested using indirect ELISA.

The study concluded that SS1 was successfully expressed in the rice plants. Of 416 regenerated plants, 164 were transgenic and exhibited normal growth in field conditions when compared to non-transgenic rice plants. One plant exhibited a high expression level and accumulated the highest amount of recombinant SS1 protein, at ~31.5 ± 1.4 ng/g dry weight (DW) grain. Others showed slightly lower expression levels, ranging from 15.8 ± 0.7 to 26.3 ± 1.1 ng/g DW grain, and five plants showed no expression.

Further Western blot analysis using antibodies against S and preS1 protein indicated that the rice-derived recombinant SS1 protein possessed both S and preS1 antigenicity. The results also confirmed that the recombinant SS1 protein could self-assemble into VLPs, which is indicative of a strong immunogenic response. Recombinant SS1 from transgenic rice induced a specific antibody response against both S and preS1 in immunized mice. Specific preS1 antibodies, which were detected four weeks after the initial antigen reaction, reached a peak value of 0.475 ± 0.055 (OD_450:630) three weeks after the final boosting injection. Specific S antibodies were first detected five weeks after the initial antigen injection, and reached a peak of 0.446 ± 0.053 (OD_450:630) in the eighth week. During subsequent weeks antibodies against both S and preS1 remained at relatively high levels. The control mice immunized with crude proteins from non-transgenic rice seeds showed no immune response to S and preS1.

In summary, the researchers expressed recombinant SS1 protein in rice seeds, which in turn induced immunological responses against S and preS1 protein in mice. From this study we can infer that the rice-derived SS1 protein could be developed as an alternative oral vaccine for preventing HBV infection in humans.

Qian B et al. (2007) Immunogenicity of recombinant hepatitis B virus surface antigen fused with preS1 epitopes expressed in rice seeds. Transgenic Research DOI 10.1007/s11248-007-9135-6 (Online First edition)

AVOIDING INSECT RESISTANCE TO CRY TOXINS FROM BACILLUS THURINGIENSIS

Mario Soberón and Alejandra Bravo

Transgenic plants as an alternative to insect control
As the world faces a skyrocketing food shortage crisis, the agricultural community is challenged with the task of increasing food production to meet this demand. Because 35% of crops are lost from pest damage due to insects, fungus, bacteria, and viruses, an efficient pest control program is an important component of any effort to increase crop yields.

Some of the chemical insecticides currently used to control insect pests are extremely toxic to non-target organisms and often deleterious to human and animal health. They pollute soils and water, since most are recalcitrant to breakdown. In addition, due to the high use of these compounds, many insects have developed resistance to different pesticides.

One alternative to chemical insecticides is the use of the Bt plants that express insecticidal proteins. In 2006, more than 32 million hectares were cultivated with Bt crops worldwide¹. The insecticidal proteins in these transgenic crops originate from the cry genes of Bacillus thuringiensis (Bt) bacteria. Cry proteins have been classified into 54 groups according to their amino acid sequence. They are highly specific; all show activity against a limited number of susceptible insects. Cry proteins are active against some lepidopteran, coleopteran, or dipteran insects, and a few are toxic to nematodes. Bt corn and Bt cotton produce the Cry1Ab and Cry1Ac proteins, respectively, active against the main lepidopteran insect pests in these crops.

Resistance to Cry toxins
The most important problem that threatens the effectiveness of Bt plants is the evolution of resistance to Bt toxins in susceptible insects. The high level and constitutive expression of Cry proteins in these plants presents a selection pressure on insect populations with increasing resistance to the toxins. Although field-evolved resistance to Bt crops has not been documented yet, laboratory strains of many pests have been selected for resistance to Bt toxins, and two different lepidopteran insects (Plutella xylostella and Trichoplusia ni, Fig. 1) have evolved resistance to Cry1Ac in Bt sprays in the field and in greenhouses, respectively^2,3.