Field performance and nitrous oxide emissions of transgenic nitrogen use efficient rice lines cultivated in tropical paddy fields.

Nitrogen (N) fertilizers make up the majority of the input used in rice production, and their excess application leads to significant environmental pollution. Developing rice varieties with improved nitrogen use efficiency (NUE) is essential to maintain the sustainability of rice production. This study aims to evaluate the performance of transgenic Oryza sativa japonica cv. Kitaake expressing the barley (Hordeum vulgare) alanine aminotransferase (HvAlaAT) gene in response to different levels of N fertilizer application under tropical paddy field conditions. Results from this study demonstrate that transgenic nitrogen use efficient Kitaake rice (Kitaake NUE) displays a grain yield increase of up to 41% compared to Kitaake null. Transgenic Kitaake NUE expressing the HvAlaAT gene displays a higher N uptake and achieves a higher nitrogen use efficiency compared to control plants while maintaining lower nitrous oxide (N2O) fluxes. The reduction in N2O emissions in Kitaake NUE compared to Kitaake null ranges from 37.5 to 96.3%. The transgenic Kitaake NUE used in this study has potential as a donor to improve the nitrogen use efficiency of indica rice for better adaptability to tropical conditions.

Protease inhibitors from several classes work synergistically against Callosobruchus maculatus.

Targeting multiple digestive proteases may be more effective in insect pest control than inhibition of a single enzyme class. We therefore explored possible interactions of three antimetabolic protease inhibitors fed to cowpea bruchids in artificial diets, using a recombinant soybean cysteine protease inhibitor scN, an aspartic protease inhibitor pepstatin A, and soybean Kunitz trypsin inhibitor KI. scN and pepstatin, inhibiting major digestive cysteine and aspartic proteases, respectively, significantly prolonged the developmental time of cowpea bruchids individually. When combined, the anti-insect effect was synergistic, i.e., the toxicity of the mixture was markedly greater than that of scN or pepstatin alone. KI alone did not impact insect development even at relatively high concentrations, but its anti-insect properties became apparent when acting jointly with scN or scN plus pepstatin. Incubating KI with bruchid midgut extract showed that it was partially degraded. This instability may explain its lack of anti-insect activity. However, this proteolytic degradation was inhibited by scN and/or pepstatin. Protection of KI from proteolysis in the insect digestive tract thus could be the basis for the synergistic effect. These observations support the concept that cowpea bruchid gut proteases play a dual role; digesting protein for nutrient needs and protecting insects by inactivating dietary proteins that may otherwise be toxic. Our results also suggest that transgenic resistance strategies that involve multigene products are likely to have enhanced efficacy and durability.

Soyacystatin N inhibits proteolysis of wheat alpha-amylase inhibitor and potentiates toxicity against cowpea weevil.

Genetic engineering may be used to introduce multiple insect resistance genes with different modes of action into crop plants. We explored the possible interactions of two differing gene products fed in the diet of cowpea weevil, Callosobruchus maculates (F.), a stored grain pest. The soybean cysteine protease inhibitor soyacystatin N (scN) and alpha-amylase inhibitor (alphaAI) from wheat have defensive function against this coleopteran. When artificial seeds containing both scN and alpha(AI) were infested with eggs of C. maculatus, the delays in larval development were longer than was predicted by summing the developmental delays seen when larvae were fed a diet containing the individual proteins, indicating that the effects of scN and alpha(AI) are synergistic. Alpha(AI) was readily hydrolyzed when incubated with insect gut extract. This proteolytic degradation was inhibited by scN, but not by Kunitz inhibitor (a serine protease inhibitor). Thus, degradation of alpha(AI) was due to proteolysis by insect digestive cysteine proteases. These data suggest that C. maculatus uses digestive enzymes not only to function in food protein digestion but also to defend the insects themselves by helping reduce the concentration of a toxic dietary protein.