Yinxiao Wang

Developing simple and efficient multi-gene expression systems is crucial for multi-trait improvement or bioproduction in transgenic plants. In previous research, an IGG6-based bicistronic system from the nonpathogenic fungus Glarea lozoyensis efficiently expressed multiple enzyme proteins in yeast and maize, and the heterologous enzymes successfully performed their catalytic activity to reconstruct the biosynthetic pathway in the host organism. Unlike enzyme proteins, some heterologous functional proteins (such as insecticidal proteins) are dose-dependent and they need to express sufficient levels to perform their biological functions. It remains unclear whether the IGG6-based bicistronic system can achieve high expression of the functional proteins for practical applications in crops. In this study, two Bacillus thuringiensis (Bt) insecticidal genes, vip3Aa and cry1Ab, were linked via IGG6 to form a bicistron, while two glyphosate resistance genes, gr79epsps and gat, served as monocistronic selectable marker genes. Regenerated maize plants were produced through genetic transformation. RNA and immunoblot analyses revealed that the vip3Aa-IGG6-cry1Ab bicistron was transcribed as a single transcript, which was then translated into two separate proteins. Notably, the transcription and translation of cry1Ab were significantly positively correlated with those of vip3Aa. Through ELISA and leaf bioassay, we identified two transgenic maize lines, VICGG-15 and VICGG-20, that exhibited high insecticidal activity against fall armyworm (FAW; Spodoptera frugiperda) and Asian corn borer (ACB; Ostrinia furnacalis), both of which had high expression of Vip3Aa and Cry1Ab proteins. Subsequent evaluations, including silk, ear, and field bioassays, as well as glyphosate tolerance assessments, indicated that the VICGG-15 plants displayed high resistance to FAW and ACB, and could tolerate up to 3600 g acid equivalent (a.e.) glyphosate per hectare without adversely affecting phenotype or yield. Our finding established that the IGG6-based bicistronic system can achieve high expression of functional proteins in maize, and it is a potential candidate for multi-gene assembly and expression in plants.

The influence of biotech crops on microbial communities in rhizosphere soil is an important issue in biosafety assessments. The transgenic maize HGK60 harboring the Bt cry1Ah gene enhanced the resistance to lepidopteran pests, while the ecological risk of HGK60 maize on rhizosphere microorganisms is unclear. In this study, we comprehensively analyzed the diversity and composition of bacterial and fungal communities in the rhizosphere soil around Bt maize HGK60 and the near-isogenic non-Bt maize ZD958 at four growth stages via a high-throughput sequencing technique. The results showed that HGK60 maize unleashed temporary effects on the bacterial and fungal diversity and richness during the study plant’s development, which would be restored after one cycle of plant cultivation due to the application of the same agricultural management. The differences of bacterial and fungal communities were marked by seasonality, while the different growth stage was the important factor as opposed to the cultivar contributing to the shifts in the bacterial and fungal communities’ structure. This study will provide useful information regarding the impact of Bt transgenic maize on the soil microbiome and a theoretical basis for the development of a safety assessment approach for Bt maize in China.

Allantoin is crucial for plant growth and development as well as adaptations to abiotic stresses, but the underlying molecular mechanisms remain unclear. In this study, we comprehensively analyzed the physiological indices, transcriptomes, and metabolomes of rice seedlings following salt, allantoin, and salt + allantoin treatments. The results revealed that exogenous allantoin positively affects the salt tolerance by increasing the contents of endogenous allantoin with antioxidant activities, increasing the reactive oxygen species (ROS)–scavenging capacity, and maintaining sodium and potassium homeostasis. The transcriptome analysis detected the upregulated expression genes involved in ion transport and redox regulation as well as the downregulated expression of many salt-induced genes related to transcription and post-transcriptional regulation, carbohydrate metabolism, chromosome remodeling, and cell wall organization after the exogenous allantoin treatment of salt-stressed rice seedlings. Thus, allantoin may mitigate the adverse effects of salt stress on plant growth and development. Furthermore, a global metabolite analysis detected the accumulation of metabolites with antioxidant activities and intermediate products of the allantoin biosynthetic pathway in response to exogenous allantoin, implying allantoin enhances rice salt tolerance by inducing ROS scavenging cascades. These results have clarified the transcript-level and metabolic processes underlying the allantoin-mediated salt tolerance of rice.

N⁶-methyladenosine (m⁶A) methylation represents a new layer of the epitranscriptomic regulation of plant development and growth. However, the effects of m⁶A on rice responses to environmental stimuli remain unclear. In this study, we performed a methylated-RNA immunoprecipitation sequencing analysis and compared the changes in m⁶A methylation and gene expression in rice under salt stress conditions. Salt stress significantly increased the m⁶A methylation in the shoots (p value < 0.05). Additionally, 2537 and 2304 differential m⁶A sites within 2134 and 1997 genes were identified in the shoots and roots, respectively, under salt stress and control conditions. These differential m⁶A sites were largely regulated in a tissue-specific manner. A unique set of genes encoding transcription factors, antioxidants, and auxin-responsive proteins had increased or decreased m⁶A methylation levels only in the shoots or roots under salt stress, implying m⁶A may mediate salt tolerance by regulating transcription, ROS homeostasis, and auxin signaling in a tissue-specific manner. Integrating analyses of m⁶A modifications and gene expression changes revealed that m⁶A changes regulate the expression of genes controlling plant growth, stress responses, and ion transport under saline conditions. These findings may help clarify the regulatory effects of m⁶A modifications on rice salt tolerance.

Gibberellin 2-oxidase (GA2ox) plays an important role in the GA catabolic pathway and the molecular function of the OsGA2ox genes in plant abiotic stress tolerance remains largely unknown. In this study, we functionally characterized the rice gibberellin 2-oxidase 8 (OsGA2ox8) gene. The OsGA2ox8 protein was localized in the nucleus, cell membrane, and cytoplasm, and was induced in response to various abiotic stresses and phytohormones. The overexpression of OsGA2ox8 significantly enhanced the osmotic stress tolerance of transgenic rice plants by increasing the number of osmotic regulators and antioxidants. OsGA2ox8 was differentially expressed in the shoots and roots to cope with osmotic stress. The plants overexpressing OsGA2ox8 showed reduced lengths of shoots and roots at the seedling stage, but no difference in plant height at the heading stage was observed, which may be due to the interaction of OsGA2ox8 and OsGA20ox1, implying a complex feedback regulation between GA biosynthesis and metabolism in rice. Importantly, OsGA2ox8 was able to indirectly regulate several genes associated with the anthocyanin and flavonoid biosynthetic pathway and the jasmonic acid (JA) and abscisic acid (ABA) biosynthetic pathway, and overexpression of OsGA2ox8 activated JA signal transduction by inhibiting the expression of jasmonate ZIM domain-containing proteins. These results provide a basis for a future understanding of the networks and respective phenotypic effects associated with OsGA2ox8.