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.

Stress responsive ZmWRKY53 gene increases cold tolerance in rice.

Plant WRKY transcription factors are responsible for biotic and abiotic stresses and play an important role in enhancing their adaptability. The AtWRKY33 is a gene that functions in response to abiotic stresses such as low temperature, drought, salinity, etc. In this study, a recombinant vector YG8198-ZmWRKY53 carrying the ZmWRKY53, an interspecific homolog of the dicotyledonous AtWRKY33, was transferred to rice plants by Agrobacterium mediated transformation. The ectopic expression of the ZmWRKY53 in transgenic rice plants conferred cold tolerance with a higher accumulation of free proline and water-soluble sugars, an increase in chlorophyll content, a decrease in electrolyte leakage rate and MDA levels compared to control plants. This result suggests that ZmWRKY53 may confer cold tolerance in rice.

Resveratrol-Enriched Rice Callus Extract Inhibits Oxidative and Cellular Melanogenic Activities in Melan-A Cells.

The excessive production of melanin can cause skin diseases and hyperpigmentation. In this study, resveratrol contained in Dongjin rice seed (DJ526) was increased through callus induction. The antioxidant capacity of resveratrol-enriched rice callus was evaluated using the ABTS radical scavenging method and was equivalent to that of vitamin C. DJ526 rice callus extract significantly increased antioxidant activities in a concentration-dependent manner. The anti-melanogenesis effects of DJ526 rice callus extract were also evaluated in melan-a cells. Resveratrol-enriched rice callus extract significantly (i) decreased the size and number of melanin-containing cells, (ii) suppressed the activity of cellular tyrosinase and melanin content, (iii) downregulated the expression of microphthalmia-associated transcription factor, tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2, (iv) increased the expression of phosphorylated extracellular signal-regulated kinase 1/2 and protein kinase B, and (v) inhibited the activation of phosphorylated p38 in melan-a cells. From the above observations, DJ526 rice callus extract showed strong antioxidant and anti-melanogenesis activity at the concentration test. These findings indicate the potential of resveratrol-enriched rice callus as a novel agent for controlling hyperpigmentation.

Melatonin-Regulated Chaperone Binding Protein Plays a Key Role in Cadmium Stress Tolerance in Rice, Revealed by the Functional Characterization of a Novel Serotonin N-Acetyltransferase 3 (SNAT3) in Rice.

The study of the mechanisms by which melatonin protects against cadmium (Cd) toxicity in plants is still in its infancy, particularly at the molecular level. In this study, the gene encoding a novel serotonin N-acetyltransferase 3 (SNAT3) in rice, a pivotal enzyme in the melatonin biosynthetic pathway, was cloned. Rice (Oryza sativa) OsSNAT3 is the first identified plant ortholog of archaeon Thermoplasma volcanium SNAT. The purified recombinant OsSNAT3 catalyzed the conversion of serotonin and 5-methoxytryptamine to N-acetylserotonin and melatonin, respectively. The suppression of OsSNAT3 by RNAi led to a decline in endogenous melatonin levels followed by a reduction in Cd tolerance in transgenic RNAi rice lines. In addition, the expression levels of genes encoding the endoplasmic reticulum (ER) chaperones BiP3, BiP4, and BiP5 were much lower in RNAi lines than in the wild type. In transgenic rice plants overexpressing OsSNAT3 (SNAT3-OE), however, melatonin levels were higher than in wild-type plants. SNAT3-OE plants also tolerated Cd stress, as indicated by seedling growth, malondialdehyde, and chlorophyll levels. BiP4 expression was much higher in the SNAT3-OE lines than in the wild type. These results indicate that melatonin engineering could help crops withstand Cd stress, resulting in high yields in Cd-contaminated fields.

Production of Mature Recombinant Human Activin A in Transgenic Rice Cell Suspension Culture.

Activin A belongs to the transforming growth factor (TGF) family member, which exhibits a wide range of biological activities, including the regulation of cellular proliferation and differentiation and the promotion of neuronal survival. The isolation of AA from natural sources can only produce limited quantities of this bioactive protein. In this study, the whole gene of the precursor form of recombinant human activin A (rhAA) contains a signal peptide, and a pro-region and a mature region were cloned into an expression vector under the control of the rice α-amylase 3D (RAmy3D) promoter. To obtain the mature (active) form of rhAA, an enterokinase cleavage site was inserted between the pro-region and mature region of rhAA. The rice seed (Oryza sativa L. cv. Dongjin) was transformed with recombinant vectors by the Agrobacterium-mediated method, and the integration of the target gene into the plant genome was confirmed by genomic PCR. The transcript expression of rhAA in transgenic rice calli was confirmed by a Northern blot analysis of mRNA. The production of rhAA was verified by Western blot analysis and ELISA. The accumulation of secreted rhAA in the culture medium was purified by Ni2+-NTA. The mature form of AA was released from the precursor form of rhAA after proteolytically processing with enterokinase. Western blot shows that the mature AA was split into monomer and homodimer with molecular weights of 14 kDa and 28 kDa under reducing and non-reducing conditions, respectively. These results suggest that the mature form of rhAA could be produced and purified using transgenic rice cell suspension culture.

A universal design of restructured dimer antigens: Development of a superior vaccine against the paramyxovirus in transgenic rice.

The development of vaccines, which induce effective immune responses while ensuring safety and affordability, remains a substantial challenge. In this study, we proposed a vaccine model of a restructured "head-to-tail" dimer to efficiently stimulate B cell response. We also demonstrate the feasibility of using this model to develop a paramyxovirus vaccine through a low-cost rice endosperm expression system. Crystal structure and small-angle X-ray scattering data showed that the restructured hemagglutinin-neuraminidase (HN) formed tetramers with fully exposed quadruple receptor binding domains and neutralizing epitopes. In comparison with the original HN antigen and three traditional commercial whole virus vaccines, the restructured HN facilitated critical epitope exposure and initiated a faster and more potent immune response. Two-dose immunization with 0.5 µg of the restructured antigen (equivalent to one-127th of a rice grain) and one-dose with 5 µg completely protected chickens against a lethal challenge of the virus. These results demonstrate that the restructured HN from transgenic rice seeds is safe, effective, low-dose useful, and inexpensive. We provide a plant platform and a simple restructured model for highly effective vaccine development.

The Dsup coordinates grain development and abiotic stress in rice.

DNA damage is a serious threat to all living organisms and may be induced by environmental stressors. Previous studies have revealed that the tardigrade (Ramazzotius varieornatus) DNA damage suppressor protein Dsup has protective effects in human cells and tobacco. However, whether Dsup provides radiation damage protection more widely in crops is unclear. To explore the effects of Dsup in other crops, stable Dsup overexpression lines through Agrobacterium-mediated transformation were generated and their agronomic traits were deeply investigated. In this study, the overexpression of Dsup not only enhanced the DNA damage resistance at the seeds and seedlings stages, they also exhibited grain size enlargement and starch granule structure and cell size alteration by the scanning electron microscopy observation. Notably, the RNA-seq revealed that the Dsup plants increased radiation-related and abiotic stress-related gene expression in comparison to wild types, suggesting that Dsup is capable to coordinate normal growth and abiotic stress resistance in rice. Immunoprecipitation enrichment with liquid chromatography-tandem mass spectrometry (IP-LC-MS) assays uncovered 21 proteins preferably interacting with Dsup in plants, suggesting that Dsup binds to transcription and translation related proteins to regulate the homeostasis between DNA protection and plant development. In conclusion, our data provide a detailed agronomic analysis of Dsup plants and potential mechanisms of Dsup function in crops. Our findings provide novel insights for the breeding of crop radiation resistance.

An accurate, reliable, and universal qPCR method to identify homozygous single insert T-DNA with the example of transgenic rice.

Early determination of transgenic plants that are homozygous for a single locus T-DNA insert is highly desirable in most fundamental and applied transgenic research. This study aimed to build on an accurate, rapid, and reliable quantitative real-time PCR (qPCR) method to fast-track the development of multiple homozygous transgenic rice lines in the T1 generation, with low copy number to single T-DNA insert for further analyses. Here, a well-established qPCR protocol, based on the OsSBE4 reference gene and the nos terminator, was optimized in the transgenic Japonica rice cultivar Nipponbare, to distinguish homozygous single-insert plants with 100% accuracy. This method was successfully adapted to transgenic Indica rice plants carrying three different T-DNAs, without any modifications to quickly develop homozygous rice plants in the T1 generation. The accuracy of this qPCR method when applied to transgenic Indica rice approached 100% in 12 putative transgenic lines. Moreover, this protocol also successfully detected homozygous single-locus T-DNA transgenic rice plants with two-transgene T-DNAs, a feature likely to become more popular in future transgenic research. The assay was developed utilizing universal primers targeting common sequence elements of gene cassettes (the nos terminator). This assay could therefore be applied to other transgenic plants carrying the nos terminator. All procedures described here use standardized qPCR reaction conditions and relatively inexpensive dyes, such as SYBR Green, thus the qPCR method could be cost-effective and suitable for lower budget laboratories that are involved in rice transgenic research.

Polyphosphate promotes oxidation resistance of ppk-expressing transgenic rice in low phosphorus culture.

Phosphorus (P) plays a crucial role in plant growth. Insufficient availability of inorganic phosphate (Pi) can significantly impact crop yields. To address this, we previously developed transgenic rice expressing the low polyphosphate kinase gene (ppk) - known as ETRS - to enhance the efficiency of P resource utilization. Previous studies have shown that ETRS thrives and presents high yields in the low P culture. ETRS and wild-type rice (WT) were cultivated to the heading stage at 15 µM of P in the low P (LP) culture and 300 µM of P in the normal culture (CK) to identify the molecular pathways behind low P tolerance. Our findings revealed that polyphosphate (polyP) significantly enhanced the growth performance of ETRS in the LP culture. This enhanced tolerance can be attributed to polyP's capacity to mitigate oxidative damage induced by LP. This was evidenced by the reduction in levels of superoxide radicals, hydrogen peroxide, and malondialdehyde. PolyP also improved the antioxidant capacity of ETRS under LP stress by regulating enzymatic antioxidants viz., superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as non-enzymatic antioxidants such as ascorbate (AsA) and glutathione (GSH). In addition, transcriptomics analysis suggested that polyP synthesis positively promoted the expressions of SOD, POD, and CAT related genes and played an active role in regulating the expression of AsA-GSH cycle system related genes in ETRS in the LP culture. These results strongly support the notion that polyP within ETRS mitigates oxidative damage through enhancement of the antioxidant system, ultimately bolstering tolerance to LP conditions.

The Synergistic Effects of Environmental and Genetic Factors on the Regulation of Anthocyanin Accumulation in Plant Tissues.

Anthocyanin accumulation is responsible for the coloration of apple fruit, and their accumulation depends on the expression of anthocyanin biosynthesis-related genes. Light is an environmental stimulus that induces fruit color by regulating genes involved in the anthocyanin biosynthesis pathway. In this study, the roles of light and genetic factors on fruit coloration and anthocyanin accumulation in apple fruit were investigated. Three genes in the anthocyanin biosynthesis pathway, MdCHS, MdANS, and MdUFGT1, were synthesized and cloned into a viral-based expression vector system for transient expression in 'Ruby S' apple fruits. Apple fruits were agroinfiltrated with expression vectors harboring MdCHS, MdANS, and MdUFGT1. Agroinfiltrated apple fruits were then either kept in the dark (bagged fruits) or exposed to light (exposed fruits). The agroinfiltrated fruits showed significantly different coloration patterns, transcript expression levels, and anthocyanin accumulation compared to the control fruits. Moreover, these parameters were higher in exposed fruits than in bagged fruits. For stable expression, MdCHS was introduced into a binary vector under the control of the rice α-amylase 3D (RAmy3D) promoter. The ectopic overexpression of MdCHS in transgenic rice calli showed a high accumulation of anthocyanin content. Taken together, our findings suggest that light, together with the overexpression of anthocyanin biosynthesis genes, induced the coloration and accumulation of anthocyanin content in apple fruits by upregulating the expression of the genes involved in the anthocyanin biosynthesis pathway.

Protopanaxadiol-Enriched Rice Exerted Antiadipogenic Activity during 3T3-L1 Differentiation and Anti-Inflammatory Activity in 3T3-L1 Adipocytes.

Ginseng is a traditional medicine with health benefits for humans. Protopanaxadiol (PPD) is an important bioactive compound found in ginseng. Transgenic rice containing PPD has been generated previously. In the present study, extracts of this transgenic rice were evaluated to assess their antiadipogenic and anti-inflammatory activities. During adipogenesis, cells were treated with transgenic rice seed extracts. The results revealed that the concentrations of the rice seed extracts tested in this study did not affect cell viability at 3 days post-treatment. However, the rice seed extracts significantly reduced the accumulation of lipids in cells and suppressed the activation of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ), which in turn inhibited the expression of adipogenesis-related mRNAs, such as adiponectin, PPARγ, C/EBPα, sterol regulatory element-binding protein 1, glucose transport member 4, and fatty acid synthase. In adipocytes, the extracts significantly reduced the mRNA expression of inflammation-related factors following LPS treatment. The activation of NF-κB p65 and ERK 1/2 was inhibited in extract-treated adipocytes. Moreover, treatment with extract #8 markedly reduced the cell population of the G2/M phase. Collectively, these results indicate that transgenic rice containing PPD may act as an obesity-reducing and/or -preventing agent.

Rice-produced classical swine fever virus glycoprotein E2 with herringbone-dimer design to enhance immune responses.

Pestiviruses, including classical swine fever virus, remain a concern for global animal health and are responsible for major economic losses of livestock worldwide. Despite high levels of vaccination, currently available commercial vaccines are limited by safety concerns, moderate efficacy, and required high doses. The development of new vaccines is therefore essential. Vaccine efforts should focus on optimizing antigen presentation to enhance immune responses. Here, we describe a simple herringbone-dimer strategy for efficient vaccine design, using the classical swine fever virus E2 expressed in a rice endosperm as an example. The expression of rE2 protein was identified, with the rE2 antigen accumulating to 480 mg/kg. Immunological assays in mice, rabbits, and pigs showed high antigenicity of rE2. Two immunizations with 284 ng of the rE2 vaccine or one shot with 5.12 µg provided effective protection in pigs without interference from pre-existing antibodies. Crystal structure and small-angle X-ray scattering results confirmed the stable herringbone dimeric conformation, which had two fully exposed duplex receptor binding domains. Our results demonstrated that rice endosperm is a promising platform for precise vaccine design, and this strategy can be universally applied to other Flaviviridae virus vaccines.

Co-expression of four penaeidins in transgenic rice seeds: an alternative strategy for substitute antibiotic agricultural products.

The co-expression of multiple antimicrobial peptides (AMPs) in genetically modified (GM) crops can give plants a broader antibacterial spectrum and lower the pathogen risk of drug resistance. Therefore, four penaeidins (shrimp-derived AMPs) were fused and encoded in an artificial gene (PEN1234), driven by the seed-specific promoter Pzein, with the aim of co-expression in seeds of transgenic rice. The resistant rice plants, acquired via Agrobacterium-mediated transformation and glufosinate screening, were identified by PCR and the modified disk-diffusion method, and eight GM lines with high AMP content in the seeds were obtained. Among them, the PenOs017 line had the largest penaeidin content, at approximately 251-300 µg/g in seeds and 15-47 µg/g in roots and leaves. The AMPs in the seeds kept their antibacterial properties even after the seed had been boiled in hot water and could significantly inhibit the growth of methicillin-resistant Staphylococcus aureus, and AMPs in the leaves could effectively inhibit Xanthomonas oryzae pv. Oryzae. The results indicate that PenOs017 seeds containing AMPs are an ideal raw-material candidate for antibiotic-free food and feed, and may require fewer petrochemical fungicides or bactericides for disease control during cultivation than conventional rice.

Use of Germination to Enhance Resveratrol Content and Its Anti-Inflammatory Activity in Lipopolysaccharide-Stimulated RAW264.7 Cells.

Inflammation is triggered by a variety of danger signals and is now a worldwide concern. Resveratrol, a natural nonflavonoid polyphenol found in naturally consumed plants and foods, has a wide spectrum of bioactive potency. We successfully generated resveratrol-enriched rice by introducing the resveratrol biosynthesis gene into Dongjin rice. In this study, resveratrol- and piceid-enriched rice (DJ526) was investigated for its anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated RAW264.7 cells compared to normal rice (DJ). In addition, the 5-day-old germinated DJ526 (DJ526_5) was tested for its anti-inflammatory effects. The piceid and resveratrol amounts increased in DJ526_5 by germination. Treatment of LPS-stimulated RAW264.7 cells with resveratrol-enriched rice seed extracts (DJ526_0 and DJ526_5) significantly decreased the production of nitric oxide (NO) and the inflammatory mediator prostaglandin E2 (PGE2), downregulated proinflammatory gene expression, and inhibited nuclear factor kappa B (NF-κB) p65, p38 mitogen-activated protein kinase, and extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation. These findings demonstrated the anti-inflammatory mechanisms of resveratrol-enriched rice in LPS-stimulated RAW264.7 cells. Furthermore, resveratrol-enriched rice could be a potential source of anti-inflammatory agents.

Phenotypic and microarray analysis reveals salinity stress-induced oxidative tolerance in transgenic rice expressing a DEAD-box RNA helicase, OsDB10.

Helicases are the motor proteins not only involved in the process of mRNA metabolism but also played a significant role in providing abiotic stresses tolerance. In this study, a DEAD-box RNA helicase OsDB10 was cloned and functionally characterized. The transcript levels of OsDB10 were increased both in shoot and root upon salt, heat, cold, and ABA application and was more prominent in shoot compared to root. Genomic integration of OsDB10 in transgenic rice was confirmed by PCR, Southern blot and qRT-PCR analysis. The transgenic plants showed quicker seed germination, reduced necrosis, higher chlorophyll, more survival rate, better seedling growth, and produced more grain yield under salinity stress. Furthermore, transgenic lines also accumulated less Na+ and high K+ ions and salinity tolerance of the transgenic were also assayed by measuring different bio-physiological indices. Moreover, the OsDB10 transgenic plants showed enhanced tolerance to salinity-induced oxidative stress by scavenging ROS and increased activity of antioxidants enzymes. Microarray analysis showed upregulation of transcriptional regulations and metabolic reprogramming as OsDB10 overexpression modulates the expression of many other genes. Altogether, our results confirmed that OsDB10 is a functional DEAD-box RNA helicase and played vital roles in plant defence response against salinity stress.

iTRAQ based proteomic analysis of rice lines having single or stacked blast resistance genes: Pi54/Pi54rh during incompatible interaction with Magnaporthe oryzae.

Deployment of single or multiple blast resistance (R) genes in rice plant is considered to be the most promising approach to enhance resistance against blast disease caused by fungus Magnaporthe oryzae. At the proteome level, relatively little information about R gene mediated defence mechanisms for single and stacking resistance characteristics is available. The overall objective of this study is to look at the proteomics of rice plants that have R genes; Pi54, Pi54rh and stacked Pi54 + Pi54rh in response to rice blast infection. In this study 'isobaric tag for relative and absolute quantification' (iTRAQ)-based proteomics analysis was performed in rice plants at 72-h post inoculation with Magnaporthe oryzae and various differentially expressed proteins were identified in these three transgenic lines in comparison to wild type during resistance response to blast pathogen. Through STRING analysis, the observed proteins were further examined to anticipate their linked partners, and it was shown that several defense-related proteins were co-expressed. These proteins can be employed as targets in future rice resistance breeding against Magnaporthe oryzae. The current study is the first to report a proteomics investigation of rice lines that express single blast R gene Pi54, Pi54rh and stacked (Pi54 + Pi54rh) during incompatible interaction with Magnaporthe oryzae. The differentially expressed proteins indicated that secondary metabolites, reactive oxygen species-related proteins, phenylpropanoid, phytohormones and pathogenesis-related proteins have a substantial relationship with the defense response against Magnaporthe oryzae. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01327-3.

SNAC3 Transcription Factor Enhances Arsenic Stress Tolerance and Grain Yield in Rice (Oryza sativa L.) through Regulating Physio-Biochemical Mechanisms, Stress-Responsive Genes, and Cryptochrome 1b.

Arsenic (As) is one of the toxic heavy metal pollutants found in the environment. An excess of As poses serious threats to plants and diminishes their growth and productivity. NAC transcription factors revealed a pivotal role in enhancing crops tolerance to different environmental stresses. The present study investigated, for the first time, the functional role of SNAC3 in boosting As stress tolerance and grain productivity in rice (Oryza sativa L.). Two SNAC3-overexpressing (SNAC3-OX) and two SNAC3-RNAi transgenic lines were created and validated. The wild-type and transgenic rice plants were exposed to different As stress levels (0, 25, and 50 µM). The results revealed that SNAC3 overexpression significantly improved rice tolerance to As stress and boosted grain yield traits. Under both levels of As stress (25 and 50 µM), SNAC3-OX rice lines exhibited significantly lower levels of oxidative stress biomarkers and OsCRY1b (cryptochrome 1b) expression, but they revealed increased levels of gas exchange characters, chlorophyll, osmolytes (soluble sugars, proteins, proline, phenols, and flavonoids), antioxidant enzymes (SOD, CAT, APX, and POD), and stress-tolerant genes expression (OsSOD-Cu/Zn, OsCATA, OsCATB, OsAPX2, OsLEA3, OsDREB2B, OsDREB2A, OsSNAC2, and OsSNAC1) in comparison to wild-type plants. By contrast, SNAC3 suppression (RNAi) reduced grain yield components and reversed the aforementioned measured physio-biochemical and molecular traits. Taken together, this study is the first to demonstrate that SNAC3 plays a vital role in boosting As stress resistance and grain productivity in rice through modulating antioxidants, photosynthesis, osmolyte accumulation, and stress-related genes expression, and may be a useful candidate for further genetic enhancement of stress resistance in many crops.

The ppk-expressing transgenic rice floating bed improves P removal in slightly polluted water.

With significant economic advantages, the plant floating bed has been widely utilized in the ecological remediation of eutrophic water because of the excessive phosphorus (P) and nitrogen discharge in China. Previous research has demonstrated that polyphosphate kinase (ppk)-expressing transgenic rice (Oryza sativa L. ssp. japonica) (ETR) can increase the P absorption capacity to support rice growth and boost rice yield. In this study, the floating beds of ETR with single copy line (ETRS) and double copy line (ETRD) are built to investigate their capacity to remove aqueous P in slightly polluted water. Compared with the wild type Nipponbare (WT) floating bed, the ETR floating beds greatly reduce the total P concentration in slightly polluted water though the ETR floating beds have the same removal rates of chlorophyll-a, NO3--N, and total nitrogen in slightly polluted water. The P uptake rate of ETRD on the floating bed is 72.37% in slightly polluted water, which is higher than that of ETRS and WT on the floating beds. Polyphosphate (polyP) synthesis is a critical factor for the excessive phosphate uptake of ETR on the floating beds. The synthesis of polyP decreases the level of free intracellular phosphate (Pi) in ETR on the floating beds, simulating the phosphate starvation signaling. The OsPHR2 expression in the shoot and root of ETR on the floating bed increased, and the corresponding P metabolism gene expression in ETR was changed, which promoted Pi uptake by ETR in slightly polluted water. The Pi accumulation further promoted the growth of ETR on the floating beds. These findings highlight that the ETR floating beds, especially ETRD floating bed, have significant potential for P removal and can be exploited as a novel method for phytoremediation in slightly polluted water.

Achieving High Expression of Cry in Green Tissues and Negligible Expression in Endosperm Simultaneously via rbcS Gene Fusion Strategy in Rice.

To allay excessive public concern about the safety of transgenic foods, and to optimize insect-resistant genes expression to delay the evolution of resistance in pests, we developed a promising strategy to fuse the GOI (gene of interest) with OsrbcS (rice small subunit of ribulose bisphosphate carboxylase/oxygenase) in transgenic rice, which acted as a carrier, driven by the OsrbcS native promoter to sequester its expression in green tissues. Using eYFP as a trial, we reported a high-level accumulation of eYFP in green tissue and almost none in the seed and root of the fused construct compared to the non-fused construct. After applying this fusion strategy in insect-resistant rice breeding, recombinant OsrbcS-Cry1Ab/Cry1Ac expressed rice plants conferred high resistance to leaffolders and striped stem borers, among which two single-copy lines possessed normal agronomic performance in the field. Specifically, Cry1Ab/Cry1Ac protein levels in single-copy construct transgenic lines ranged from 1.8 to 11.5 µg g-1 in the leaf, higher than the Actin I promoter-driven control, T51-1, about 1.78 µg g-1 in the leaf, but negligible (only 0.00012-0.00117 µg g-1) in endosperm by ELISA analysis. Our study provided a novel approach to creating Cry1Ab/Cry1Ac-free endosperm rice with a high level of insect-resistant protein in green tissues through the simultaneous usage of the OsrbcS promoter and OsrbcS as a fusion partner.