Flavonoid localization in soybean seeds: Comparative analysis of wild (Glycine soja) and cultivated (Glycine max) varieties.

Wild soybean (Glycine soja) is known for its high flavonoid contents, yet the distribution of flavonoids in the seeds is not well understood. Herein, we utilized matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and metabolomics methods to systematically investigate flavonoid differences in the seed coats and embryos of G. soja and G. max. The results of flavonoid profiles and total flavonoid content analyses revealed that flavonoid diversity and abundance in G. soja seed coats were significantly higher than those in G. max whereas the levels were similar in embryos. Specifically, 23 unique flavonoids were identified in the seed coats of G. soja, including procyanidins, epicatechin derivatives, and isoflavones. Using MALDI-MSI, we further delineated the distribution of the important flavonoids in the cotyledons, hypocotyls, and radicles of the two species. These findings imply that G. soja holds considerable breeding potential to enhance the nutritional and stress resistance traits of G. max.

Seed coat colour and structure are related to the seed dormancy and overwintering ability of crop-to-wild hybrid soybean.

The possible persistence of genetically modified (GM) crop-to-wild hybrid seeds in the soil seed bank is a major concern in risk assessment and is closely related to seed characteristics such as dormancy. In the present study, we generated F3 hybrids via crosses between GM soybean accessions and wild soybean and evaluated the dormancy, overwintering ability and inheritance of foreign genes in different-coloured hybrid seeds (yellow, green, brown and black). The results revealed that the 5-enolpyruvylshikimate-3-phosphate synthase transgene may have no influence on crop wild hybrid seed dormancy and overwintering ability, and the dormancy of the hybrid seeds was closely related to seed coat colour. F3 hybrid seeds with light colours (yellow and green) were relatively nondormant, while seeds that were dark (brown and black) in colour were relatively dormant. Moreover, the hybrid seeds that were dark in colour had a much stronger overwintering ability than the lighter-coloured seeds, with 21.33 % of the black seeds and 33.33 % of the brown seeds remaining viable after 240 days of soil burial. In contrast, almost all the F3 yellow and green seeds were no longer viable during winter. Scanning electron microscopy revealed that the lighter-coloured seeds had a thin palisade layer and very few surface deposits, while the darker-coloured seeds had a thicker palisade layer and a large area of honeycomb-like surface deposits similar to those of wild soybean seeds. Thus, the physical dormancy and overwintering ability of the darker-coloured seeds may be related to the seed coat. Our results suggest that transgenes of GM soybean might disperse into wild populations and persist in seed banks.

Fitness changes in wild soybean caused by gene flow from genetically modified soybean.

BACKGROUND: Crop-wild hybridization has generated great concerns since gene flow can be an avenue for transgene escape. However, a rather limited number of studies on risk assessment regarding the dispersion of transgenes from GM soybean to populations of its wild relatives have been previously conducted. RESULTS: The results of the 3-year experiment demonstrated that hybrids between GM soybeans and wild soybean had lower seed germination and higher seed productivity than GM soybean. Both of these features of hybrid (especially F2 and F3) were similar to those of wild soybean. Furthermore, the foreign protein was stably expressed in hybrid EPSPS positive plants; however, no difference was observed in agronomic measurements between hybrids that are glyphosate sensitive or resistant, homozygous or heterozygous for the transgene, indicating that the presence of the EPSPS transgene does not affect the vigor of hybrid. In contrast, hybridization between GM soybean and wild soybean may have more impact on hybrid growth and fecundity, this increase in biomass and yield confers a potential competition benefit to hybrids. CONCLUSIONS: Gene flow from GM soybean to wild soybean has the potential to promote the adaptability of hybrids and may increase the possibility of dispersal of transgenes in wild soybean relatives.

In Situ Proteomic Analysis of Herbicide-Resistant Soybean and Hybrid Seeds via Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging.

Transgenic soybean is the commercial crop with the largest cultivation area worldwide. During transgenic soybean cultivation, exogenous genes may be transferred to wild relatives through gene flow, posing unpredictable ecological risks. Accordingly, an environmental risk assessment should focus on fitness changes and underlying mechanisms in hybrids between transgenic and wild soybeans (Glycine soja). Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) was used for in situ detection and imaging of protein changes in the seeds of transgenic herbicide-resistant soybean harboring epsps and pat genes, non-transgenic soybean, wild soybean, and their F2 hybrid. Protein data clearly distinguished wild soybeans, while the F2 seeds had protein characteristics of both parents and were distinguished from wild soybean seeds. Using UPLC-Q-TOF-MS, 22 differentially expressed proteins (DEPs) were identified, including 13 specific to wild soybean. Sucrose synthase and stress response-related DEPs were differentially expressed in parental and offspring. Differences in these may underpin the greater adaptability of the latter. MSI revealed DEP distribution in transgenic, wild, and F2 seeds. Identifying DEPs related to fitness may elucidate mechanisms underlying fitness differences among the studied varieties. Our study shows that MALDI-MSI has the potential to become a visual method for transgenic soybean analysis.

Fitness and Ecological Risk of Hybrid Progenies of Wild and Herbicide-Tolerant Soybeans With EPSPS Gene.

Exogenous genes of transgenic crops are usually transferred to their wild-type relatives through pollen-mediated gene flow, which may change the ecological fitness and ability to invade wild populations, resulting in the weeding of wild plants and other unpredictable environmental impacts. In this study, the F1 generation of herbicide-resistant soybeans and wild soybeans was obtained by artificial pollination, F2 generation seeds were obtained by self-crossing, and the fitness of the parents and their F1 and F2 generations were tested. The foreign protein EPSPS was expressed normally in the hybrid between transgenic and wild soybeans; however, the protein expression was significantly lower than that in transgenic soybeans. The fitness of the F1 hybrid between transgenic and wild soybeans was significantly lower than that of its parent. Compared with those of the wild soybeans, the F2 generation soybeans improved in some fitness indices, while the emergence rate, pollen germination rate, and number of full seeds per pod, pods per plant, and full seeds per plant did not significantly differ. The aboveground biomass and 100-seed weight of the F2 generation were higher than those of wild soybeans. Fitness among the F2-negative plants, homozygous, and heterozygous positive plants did not significantly vary. Improved fitness and presence of foreign genes in the F2 soybean were not significantly correlated. As the F2 generation of transgenic and wild soybeans had no fitness cost and the flowering stage were overlapped, the foreign gene might still spread in the wild soybean population.

Cellular Localization of Exogenous Cry1Ab/c and its Interaction with Plasma Membrane Ca2+-ATPase in Transgenic Rice.

The cellular localization of exogenous proteins expressed in transgenic crops not only determines their stability, but also their effects on crop growth and development, including under stressful conditions; however, the underlying molecular mechanisms remain unknown. Here, we determined the cellular distribution of exogenously expressed Cry1Ab/c protein in insect-resistant transgenic rice Huahui-1 (HH1) cells through subcellular localization, immunohistochemistry, immunofluorescence, and western blot analyses. Interaction between the Cry1Ab/c protein and the preliminarily screened endogenous plasma membrane protein Ca2+-ATPase was investigated through yeast two-hybrid, bimolecular fluorescence complementation (BIFC), and co-immunoprecipitation analyses. The potential interaction mechanism was analyzed by comparing the cellular localization and interaction sites between Cry1Ab/c and Ca2+-ATPase. Phenotypic indices and Ca2+-ATPase activity, which may be regulated by the Cry1Ab/c-Ca2+-ATPase interaction, were determined in transgenic HH1 and the parental line Minghui-63 under stress-free and salt-stress conditions. The results showed that Cry1Ab/c was not only distributed in the cytoplasm and nucleus but was also distributed on the plasma membrane, where it interacted with plasma membrane Ca2+-ATPase. This interaction partially retain plasma membrane protein Ca2+-ATPase in the nucleus by a BIFC experiment and thus may affect Ca2+-ATPase activity on the membrane by altering the cellular location of the protein. Consistently, our results confirmed that the presence of Cry1Ab/c in the transgenic HH1 resulted in a reduction in Ca2+-ATPase activity as well as causing detrimental effects on plant phenotype, including significantly reduced plant height and biomass, compared to parental MH63; and that these detrimental effects were more pronounced under salt stress conditions, impacting the salt resistance of the transgenic plants. We suggest that the Cry1Ab/c-Ca2+-ATPase interaction may explain the plasma membrane localization of Cry1Ab/c, which lacks a signal peptide and a transmembrane domain, and the adverse effects of Cry1Ab/c expression on the growth and development of transgenic HH1 plants under salt stress. This information may clarify the molecular mechanisms of these unintended effects and demonstrate the feasibility of evaluating the success and performance of genetic modification of commercially vital crops.

Fitness of Insect-resistant transgenic rice T1C-19 under four growing conditions combining land use and weed competition.

Transgene escape into natural ecosystems through seed spraying or transgene introgression may potentially cause environmental biosafety problems. In this study, we assessed the environmental risk of insect-resistant transgenic rice entering farmland margins or natural ecosystems adjacent to farmland. Transgenic Cry1C* rice (T1C-19) was used to study the effects of exogenous Cry1C* expression on vegetative and reproductive growth indices under different growing conditions using the following four combined treatments of land use and weeds: farmland and uncultivated land without weeds (F-NW and U-NW, respectively), and farmland and uncultivated land with weeds (F-W and U-W, respectively). The expression of Cry1C* protein under the U-NW, F-W, and U-W conditions was significantly lower than under the control condition, F-NW. Tiller number, biomass, filled grain number, filled grain weight, and other vegetative and reproductive indices were significantly lower in the rice line TIC-19 than in MH63 under F-NW and U-NW conditions, indicating a significant fitness cost. However, under F-W and U-W conditions, vegetative growth indices such as plant height, tiller number, and biomass, as well as reproductive growth indices such as filled grain number per plant, filled grain weight per plant, and seed setting rate in TIC-19 were similar to those in MH63, indicating a long-term coexistence. These results indicate a lower ecological risk of T1C-19 compared to MH63 under F-NW and U-NW, although their long-term coexistence may lead to potential ecological risks under F-W and U-W.

Expression of Bt Protein in Transgenic Bt Cotton Plants and Ecological Fitness of These Plants in Different Habitats.

Fitness is one of the key parameters to evaluate the effects of transgenic plants on the ecological environment. To evaluate the ecological risk of transgenic Bt cotton plants growing in different habitats, we determined the expression of the exogenous Bt gene and the fitness of transgenic and non-transgenic cotton plants in three habitats (farmland, grassland, and shrub). We observed that the expression of Bt protein in the farmland was significantly higher than that in the natural habitat, and when the growth environment was suitable, the Bt protein expression level showed a downward trend with the advancement of the growth. There were no significant differences in plant height, aboveground biomass, and seed yield between the Bt transgenic and non-transgenic cotton plants at the same growth stage under the same habitat. Nevertheless, in different habitats, the fitness of the same cotton line showed significant differences. In the farmland habitat, the plant height, aboveground biomass, and seed yield of both transgenic cotton and its non-transgenic isoline were significantly higher than that in the other two natural habitats. The results indicate that the expression of Bt protein does not increase the fitness of the parent plants and would not cause the weeding of the recipient cotton plants.

[Impacts of herbicide-resistant soybean ZUTS-33 with g10-epsps gene on field biodiversity]. / 转g10-epspsåŸºå› è€é™¤è‰å‰‚å¤§è±†ZUTS-33å¯¹å†œç”°ç”Ÿç‰©å¤šæ ·æ€§çš„å½±å“.

Evaluating the impacts of genetically modified crops on biodiversity is a necessary step before their release to the field and obtaining environmental safety certificates. To assess the ecological safety of herbicide-resistant soybean ZUTS-33, we compared arthropod diversity, diseases occurrence, nodule number, and weed diversity through spraying herbicide or water on ZUTS-33, and its parental control receptor HC-3 and main cultivar soybean ZH-13 in a field experiment. The results showed that there was no significant difference of arthropod diversity (number of insects per 100 plants, Shannon index, Simpson index and Pielou index), diseases incidence rates and disease index, nodules and weed diversity between ZUTS-33 and non-genetically modified control soybean HC-3 and ZH-13. Spraying herbicide on ZUTS-33 had no significant effect on arthropod diversity, diseases and rhizobium compared with those treatments of spraying clear water on ZUTS-33, non-genetically modified control HC-3 and ZH-13, and the abundance of weeds were significantly decreased.

CRISPR/Cas9 mediated genome editing of Helicoverpa armigera with mutations of an ABC transporter gene HaABCA2 confers resistance to Bacillus thuringiensis Cry2A toxins.

High levels of resistance to Bt toxin Cry2Ab have been identified to be genetically linked with loss of function mutations of an ABC transporter gene (ABCA2) in two lepidopteran insects, Helicoverpa armigera and Helicoverpa punctigera. To further confirm the causal relationship between the ABCA2 gene (HaABCA2) and Cry2Ab resistance in H. armigera, two HaABCA2 knockout strains were created from the susceptible SCD strain with the CRISPR/Cas9 genome editing system. One strain (SCD-A2KO1) is homozygous for a 2-bp deletion in exon 2 of HaABCA2 created by non-homologous end joining (NHEJ). The other strain (SCD-A2KO2) is homozygous for a 5-bp deletion in exon 18 of HaABCA2 made by homology-directed repair (HDR), which was produced to mimic the r2 resistance allele of a field-derived Cry2Ab-resistant strain from Australia. Both knockout strains obtained high levels of resistance to both Cry2Aa (>120-fold) and Cry2Ab (>100-fold) compared with the original SCD strain, but no or very limited resistance to Cry1Ac (<4-fold). Resistance to Cry2Ab in both knockouts is recessive, and genetic complementary tests confirmed Cry2Ab resistance alleles are at the same locus (i.e. HaABCA2) for the two strains. Brush border membrane vesicles (BBMVs) of midguts from both knockout strains lost binding with Cry2Ab, but maintained the same binding with Cry1Ac as the SCD strain. In vivo functional evidence from this study demonstrates knockout of HaABCA2 confers high levels of resistance to both Cry2Aa and Cry2Ab, confirming that HaABCA2 plays a key role in mediating toxicity of both Cry2Aa and Cry2Ab against H. armigera.

Resistance to Bacillus thuringiensis toxin Cry2Ab and survival on single-toxin and pyramided cotton in cotton bollworm from China.

Evolution of Helicoverpa armigera resistance to Bacillus thuringiensis (Bt) cotton producing Cry1Ac is progressing in northern China, and replacement of Cry1Ac cotton by pyramided Bt cotton has been considered to counter such resistance. Here, we investigated four of the eight conditions underlying success of the refuge strategy for delaying resistance to Cry1Ac+Cry2Ab cotton, a pyramid that has been used extensively against H. armigera outside China. Laboratory bioassays of a Cry2Ab-selected strain (An2Ab) and a related unselected strain (An) reveal that resistance to Cry2Ab (130-fold) was nearly dominant, autosomally inherited, and controlled by more than one locus. Strong cross-resistance occurred between Cry2Ab and Cry2Aa (81-fold). Weaker cross-resistance (18- to 22-fold) between Cry2Ab and Cry1A toxins was also present and significantly increased survival of An2Ab relative to An on cotton cultivars producing the fusion protein Cry1Ac/Cry1Ab or Cry1Ac. Survival on Cry1Ac+Cry2Ab cotton was also significantly higher in An2Ab than in An, showing that redundant killing on this pyramid was incomplete. Survival on non-Bt cotton did not differ significantly between An2Ab and An, indicating an absence of fitness costs affecting this trait. These results indicate that a switch to three-toxin pyramided cotton could be valuable for increasing durability of Bt cotton in China.