Precise insertion of DNA sequences at targeted locations in plant genomes is pivotal for synthetic biology, genetics, and crop improvement. Construct design plays a critical role in achieving precise insertions, yet practical guidance remains limited. This review provides an in-depth overview of construct design principles and targeted DNA insertion (knock-in) strategies in plants. We assess the strengths, limitations, and construct requirements of current knock-in methods for specific applications, including short, large, and multifragment insertions. Additionally, we explore the potential of adopting advanced nonplant technologies to enhance knock-in efficiency and precision in plants. This review provides a valuable resource for facilitating the effective application of knock-in technologies to genetically improve crops with minimal off-target effects.

To address low nitrogen removal efficiency in aquaculture tailwater with a low carbon-to-nitrogen ratio, this study constructed a multi-compartment aerobic denitrification reactor (MPBR) integrating nitrification, slow-release carbon, anoxic denitrification, and simultaneous nitrification-denitrification zones. The synergistic effects of temperature (15 °C, 25 °C), hydraulic retention time (HRT; 4 h, 8 h, 10 h), and influent NO₃⁻-N load (20 mg/L, 50 mg/L) on nitrogen removal were investigated, along with microbial community dynamics via high-throughput sequencing. The partitioned structure ensured robust nitrification, with ammonia removal exceeding 90% under all conditions. Denitrification was highly sensitive to multi-parameter interactions: low temperature (15 °C) reduced NO₃⁻-N removal from 89.4% (25 °C) to 38.1%; high load (50 mg/L) increased absolute removal when HRT was sufficient (≥8 h) but caused inhibition when HRT was insufficient; HRT exhibited a threshold (∼8 h), with limited benefit at low temperatures. The simultaneous nitrification-denitrification zone maintained effluent NO₂⁻-N below 0.2 mg/L across all conditions. Pseudomonadota dominated the microbial community (75% average abundance), correlating positively with denitrification efficiency. At the genus level, Pseudomonas thrived at room temperature, whereas Simplicispira enriched significantly in the slow-release carbon zone under low temperature (up to 47.5%), exhibiting complementary succession and sustaining denitrification potential. Spatial niche differentiation across functional zones and functional compensation under stress supported system stability. This study elucidates the multi-factor regulation of aerobic denitrification and microbial compensation mechanisms, providing a theoretical basis for applying partitioned reactors in aquaculture tailwater treatment.

Peatlands, vital global carbon sinks dominated by Sphagnum, are increasingly threatened by water table decline (WTD) and nitrogen-phosphorus (N-P) enrichment. Sphagnum relies on complex microbial communities for ecosystem functions, but the combined impacts of these stressors on its physiology, soil biogeochemistry, and microbiome dynamics were poorly understood. In this study, a controlled experiment combining WTD and N-P addition was conducted to investigate how these perturbations alter plant–soil–microbe interactions. Under WTD, soil carbon to nitrogen (C/N) and carbon to phosphorus (C/P) ratios increased while the N/P ratio decreased, indicating altered stoichiometric characteristics. Soil NH4+ concentrations increased whereas NO3− decreased. Soil microbial α-diversity declined, and community assembly analyses further indicated that dispersal limitation played a dominant role in shaping community composition. In contrast, the endophytic microbiome of Sphagnum palustre maintained relatively high network complexity, reflected by increased edge number and average degree. N–P addition increased soil total nitrogen (TN) and total phosphorus (TP) contents and partially restored soil microbial diversity, but reduced the complexity of endophytic microbial networks. Functional gene analysis showed that WTD increased the abundance of several key genes associated with carbon and nitrogen cycling (e.g., nirK, nirS), whereas N–P addition suppressed a substantial subset of these genes. Co-occurrence network analysis revealed keystone taxa shifts and links between microbial network stability, modularity, and capitulum moisture and stoichiometry (e.g., TC/TP, TN/TP). Our findings highlight a stoichiometry-driven mechanism by which hydrological and nutrient perturbations reshape plant–microbe interactions, providing new insight into peatland resilience under global change.

CRISPR-Cas12a integrated with nanomaterials has formulated powerful biosensors for viral protein detection, addressing the urgent need for point-of-care diagnostics. However, existing platforms are hindered by either multi-step separation procedures or insufficient signal amplification, limiting their sensitivity and practicality. Here, we report a one-pot "on-off" biosensor that combines metal-enhanced fluorescence (MEF) and nanoscale spatial confinement by co-localizing both reporter substrates and the CRISPR-Cas12a system on gold-silica core-shell nanoparticles (Au@SiO2 NPs), enabling rapid and ultrasensitive protein detection. Using SARS-CoV-2 nucleocapsid (N) protein as a model analyte, Au@SiO2 NPs are co-functionalized with (i) ssDNA activators blocked by N protein-specific aptamers, (ii) light-up hairpin DNA (DAP) complexed with auramine O (AO) as reporters, and (iii) short polyethylene glycol (PEG) spacers to mitigate steric hindrance. The nanoplatform displays an ultrabright "on-state" fluorescence, with an intensity >860-fold higher than that of free AO, enabled by the interaction with DAP and optimized fluorophore-metal spacing (∼20 nm). Upon target binding, aptamer displacement exposes the activator to locally initiate Cas12a trans-cleavage, disrupting proximal DAP structure and its interaction with AO, thereby producing a distinct "off-state" signal. Within the linear detection range, the system demonstrates up to ∼85% signal reduction relative to the initial signal and a signal-to-noise ratio (SNR) of 83.89, corresponding to a ∼2.5-fold higher SNR than the solution-phase system. The platform attains a limit of detection at 67.2 fg/mL within 30 min, with excellent sensitivity, selectivity, stability, and recovery in bronchoalveolar lavage fluid. By combining MEF-driven signal amplification with surface-confined CRISPR-Cas12a trans-cleavage, this platform establishes an efficient strategy for sensitive N protein detection.

Tunnel-Greenhouse aquaculture (TGA) system as a cost-effective aquaculture system has been widely and rapidly promoted in China, accounting for approximately 20 % of shrimp production area. However, the environmental impact potential of TGA is still unknown compared with conventional shrimp aquaculture systems. A life cycle assessment (LCA) methodology was applied to characterize the environmental performance and potential improvement opportunities related to TGA and conventional systems, and a questionnaire survey of 52 farmers was carried out in the coastal region of eastern China. SimaPro 9.5.0.0 software was utilized to create a cradle-tofarmgate LCA based on trial and survey data, and CML-IA techniques were employed to assess the environmental performance. The results indicated that each ton of fresh shrimp produced applying the TGA farming system generates around 4989.5 kg CO2 eq., 26.16 kg SO2 eq., and 48.46 kg PO4 eq. It also clearly demonstrated that the primary causes of environmental effects in the TGA shrimp farming system are the power used to run the farm, the usage of fishmeal as feed production raw materials, and the discharge of farming wastewater. Meanwhile, it pointed out that improving energy efficiency and reducing the use of fishmeal in feed are fundamental ways of minimizing environmental effects. Our findings could assist governments and businesses in making better decisions and support the more sustainable development of shrimp farming in China.

We review recent advances in the design of materials that are biomass-derived, performance-advantaged, and both inspired and enabled by biological approaches.

Single-cell RNA sequencing (scRNA-seq) has transformed transcriptomic studies by enabling gene expression profiling at the resolution of individual cells within and across a broad range of tissue types, revealing cellular heterogeneity that is obscured in bulk tissue transcriptomes. Over the past decade, improvements in microfluidics and library preparation have drastically increased throughput, allowing tens of thousands of cells to be assayed in a single experiment. Although initially developed in animal systems, scRNA-seq has rapidly emerged as a powerful and widely adopted approach in plant biology. Beyond transcriptomics, the integration of single-cell data with chromatin accessibility, proteomics, metabolomics, and spatial omics is enabling a system-level understanding of plant gene regulation and cellular organization. Network-based analytical frameworks further support the reconstruction of gene regulatory networks and the interpretation of complex single-cell data. In this review, we summarize the current technological landscape of plant single-cell studies, discuss key experimental and analytical challenges, and review emerging strategies for validating single-cell discoveries. We also discuss future directions in applying single-cell technologies to woody perennials plants and bioenergy-relevant crops, emphasizing their potential to accelerate the discovery of cell type-specific regulatory mechanisms underlying growth, stress resilience, and biomass production.

The treatment of high-salinity, low-carbon marine aquaculture wastewater poses significant challenges for biological denitrification. This study systematically evaluated the performance of a polycaprolactone (PCL)-based aerobic denitrification biofilter under varying temperatures (15 degrees C and 25 degrees C) and PCL addition levels (282, 564, 846, 1128, and 1410 g). Optimal nitrogen removal, total nitrogen (TN) removal efficiency exceeding 92%, was achieved with 1128 g PCL at 15 degrees C (HRT 10 h) and 1410 g PCL at 25 degrees C (HRT 8 h), significantly outperforming the low-PCL baseline treatment. Microbial community analysis revealed that increased PCL dosage promoted the dominance of the hydrolytic genus Flavobacterium over Simplicispira, enhancing polymer degradation capacity and system stability. Metagenomic sequencing further elucidated the complete PCL degradation pathway, wherein hydrolysis products were oxidized to generate NADH and FADH2, serving as electron donors for denitrification. Key functional genes (narG, nirK, nosZ) and enzymes associated with both PCL decomposition and nitrate reduction were significantly enriched in high-performance reactors (e.g., AT15H6, AT25H6, ET15H10, ET25H10), correlating strongly with observed nitrogen removal rates. By integrating reactor performance with microbial ecology and functional genetics, this work provides a comprehensive "material-microorganism-gene-performance" framework, offering both practical strategies and mechanistic insights for enhancing denitrification in saline aquaculture systems.

Geosmin (GSM) and 2-methylisoborneol (2-MIB) are the primary odorants responsible for the earthy smell in freshwater fish, which severely restricts aquaculture development. To clarify the distribution and accumulation patterns of GSM and 2-MIB in freshwater aquaculture water bodies and in major cultured species in the Tianjin area, and to provide a scientific basis for optimizing water suspension processes, this study conducted systematic sampling in September 2025. We sampled water and major cultured species—carp, silver carp, bighead carp, and largemouth bass—at 15 representative sites (3 reservoirs, 7 earthen ponds, and 5 cement slope-protection ponds). GSM and 2-MIB were quantified by purge-and-trap gas chromatography–mass spectrometry. Principal component analysis (PCA) was used to explore differences in environmental drivers and species-specific accumulation. The detection rates of GSM and 2-MIB in aquaculture pond water were 80.00% and 73.33%, respectively, and their concentration ranges were 9.77–49.99 ng/L and 10.66–71.57 ng/L, respectively; these values were significantly higher than those measured in reservoir water. PCA indicated that total nitrogen, total phosphorus, and chlorophyll a are the key environmental factors driving accumulation of off-flavor substances in water bodies. Concentrations of GSM and 2-MIB in carp muscle ranged from 51.04 to 409.97 ng/kg and from 121.37 to 808.98 ng/kg, respectively, and they were significantly positively correlated with the corresponding concentrations in the ambient water. GSM and 2-MIB levels ranked from highest to lowest as follows: silver carp, bighead carp, mirror carp, common carp, crucian carp, channel catfish, snakehead, and largemouth bass. PCA clustering further verified that feeding habits, occupied water layer, and fat content synergistically influenced differences in accumulation. Based on these results, we recommended a differentiated strategy for the purging process. This study provided a theoretical basis and data support for controlling earthy-muddy odor in freshwater fish and for precise optimization of the purging process.

Crassulacean acid metabolism (CAM) is a specialized photosynthetic pathway that enhances water-use efficiency by temporally separating nocturnal CO2 uptake from daytime decarboxylation and carbon fixation. To uncover the regulatory mechanisms coordinating these temporal dynamics, we generated high-resolution, 48 h time-course transcriptomes for the CAM model Kalanchoe fedtschenkoi under both 12 h/12 h light/dark (LD) cycles and continuous light (LL). A rhythmicity analysis revealed that diel light cues are the dominant driver of transcript oscillations: 16,810 genes (54.3% of annotated genes) exhibited rhythmic expression only under LD, whereas just 399 genes (1.3%) remained rhythmic under LL. A smaller set of 3009 genes (9.7%) oscillated in both conditions, indicating that the intrinsic circadian clock sustains rhythmicity for a limited subset of the transcriptome. A gene co-expression network analysis revealed extensive integration between circadian clock components, core CAM pathway enzymes, and stomatal regulators, defining regulatory modules that coordinate metabolic and physiological timing. Notably, key hub genes associated with post-translational and post-transcriptional regulation, including the E3 ubiquitin ligase HUB2 and several pentatricopeptide repeat (PPR) proteins, act as central nodes in CAM-associated networks. This discovery implicates epigenetic and organellar regulation as previously unrecognized critical tiers of control in CAM. Together, our results support a regulatory model in which CAM rhythmicity is governed by both external light/dark cues and the endogenous circadian clock through multi-level control spanning transcriptional and protein-level regulation. To support community exploration, we also provide an interactive eFP (electronic Fluorescent Pictograph) browser for visualizing time-resolved gene expression profiles.

For decades, Agrobacterium tumefaciens-mediated plant transformation has played an integral role in advancing fundamental and applied plant biology. The recent omnipresent emergence of synthetic biology, which relies on plant transformation to manipulate plant DNA and gene expression for novel product biosynthesis, has further propelled basic as well as applied interests in plant transformation technologies. The strong demand for a faster design-build-test-learn cycle, the essence of synthetic biology, is, however, still ill-matched with the long-standing issues of high tissue culture recalcitrance and low transformation efficiency of a wide range of plant species especially food, fiber and energy crops. To maximize the utility of plant material and improve the transformation productivity per unit plant form, we studied the regeneration and transformation efficiency of different types of explants, including leaf, stem, petiole, and root from Populus, a woody perennial bioenergy crop. Our results show that root explants, in addition to the above-ground tissues, have considerable regeneration capacity and amenability to A. tumefaciens and, the resulting transformants have largely comparable morphology, reporter gene expression, and transcriptome profile, independent of the explant source tissue. Transcriptome analyses mapped to regeneration stages and transformation efficiencies further revealed the expression of the auxin and cytokinin signaling and various developmental pathway genes in leaf and root explants undergoing early organogenesis. We further report high-potential candidate genes that may potentially be associated with higher regeneration and transformation efficiency. Overall, our study shows that explants from above- and belowground organs of a Populus plant are suitable for genetic transformation and tissue culture regeneration, and together with the underlying transcriptome data open new routes to maximize plant explant utilization, stable transformation productivity, and plant transformation efficiency.

RNA plays a central role in plants, governing various cellular and physiological processes. Monitoring its dynamic abundance provides a discerning understanding of molecular mechanisms underlying plant responses to internal (developmental) and external (environmental) stimuli, paving the way for advances in plant biotechnology to engineer crops with improved resilience, quality and productivity. In general, traditional methods for analysis of RNA abundance in plants require destructive, labour-intensive and time-consuming assays. To overcome these limitations, we developed a transformative innovation for in vivo RNA imaging in plants. Specifically, we established a synthetic split ribozyme system that converts various RNA signals to orthogonal protein outputs, enabling in vivo visualisation of various RNA signals in plants. We demonstrated the utility of this system in transient expression experiments (i.e., leaf infiltration in Nicotiana benthamiana) to detect RNAs derived from transgenes and tobacco rattle virus, respectively. Also, we successfully engineered a split ribozyme-based biosensor in Arabidopsis thaliana for in vivo visualisation of endogenous gene expression at the cellular level, demonstrating the feasibility of multi-scale (e.g., cellular and tissue level) RNA imaging in plants. Furthermore, we developed a platform for easy incorporation of different protein outputs, allowing for flexible choice of reporters to optimise the detection of target RNAs.

Main text The bioeconomy represents an advanced economic paradigm that builds upon previous agricultural,industrial,and digital economic models.It seeks to tackle critical global challenges such as resource scarcity,escalating healthcare demands,and environmental degrada-tion.

Gene stacking, the process of introducing multiple genes into a single plant to enhance desired traits, is essential for plant genetic improvement through both conventional breeding and genetic transformation. In general, transformation-based gene stacking can be achieved through either co-transformation to simultaneously introduce multiple genes or sequential multi-round transformation. While co-transformation is generally faster and more efficient than sequential multi-round transformation, it often requires two selectable marker genes, which confer resistance to antibiotics, for selecting transgenic events. However, in most cases, there is only one best selectable marker gene for a specific plant species or genotype. Also, it is harder to optimize the concentrations of two antibiotics for co-transformation than using one antibiotic for selecting transgenic events. To overcome this challenge, we recently developed an innovative split selectable marker system for plant co- transformation, allowing the use of one selectable marker gene to select transgenic events. This method involves constructing two binary vectors, each carrying a subset of genes of interest and a partial fragment of the selectable marker gene, which is connected to a partial intein fragment. Following Agrobacterium-mediated co- transformation, plants harboring both binary vectors are selected using a single antibiotic, such as kanamycin. This split-marker system can be used to co-transform multiple genes into both herbaceous and woody plants, accelerating genetic improvement of polygenic traits or integrative improvement of multiple traits to simultaneously increase crop yield and quality.

The field of synthetic biology is essential to the continued development of a bio-based economy, creating mechanisms to supply carbon needed in the economy by both converting existing end-of-life wastes as well as by creating novel, purpose-grown and sustainable feedstocks. Here, we first discuss the near- and long-term resources available for use as feedstocks for bioconversion as well as the output molecules needed for building the foundation of an expanded bio-based economy. We then outline the organisms and phenotypic traits that are needed for the performance-advantaged chassis organisms of the future. Furthermore, we detail the advances, challenges, and opportunities in both microbial and plant synthetic biology relevant to expanding the bio-based economy. Finally, we explore technologies that have and will further enable advances in synthetic biology and the greater bio-based economy.

Aerobic denitrification, a novel method for complete nitrate removal process, relies on a constant carbon supply, and carbon availability can be a limiting factor for the process. Three biodegradable polymers - polybutylene succinate (PBS), polycaprolactone (PCL), and polylactic acid (PLA) were used as carbon source of aerobic denitrifying bacteria, Halomonas venusta for treating recirculating aquaculture wastewater in batch tests, respectively. The group utilizing PCL displayed the greatest efficiency in nitrate removal. Afterwards, a continuous flow airlift fluidized bed (CAFB) - aerobic denitrification bioreactor using PCL(PADR) as bio-carriers and carbon source was started up. The effect of shifting temperature on the nitrate removal and microbial community of CAFB-PADR was investigated under different hydraulic retention times (HRT). The results showed that no significant difference was detected in denitrification activity at 25 and 30 degrees C condition, where the denitrification rate was 1.1-1.8 times higher than that at 15 degrees C. However, the nitrogen removal efficiency was above 50 % and there was barely accumulation of nitrite at 15 degrees C, indicating the good nitrate removal performance in CAFB-PADR at all three temperature conditions. Microbial composition analyses revealed that as the temperature decreased, the relative abundance of Gammaproteobacteria increased, while those of Alphaproteobacteria and Bacteroidia diminished decreased. The existence of denitrifying function genera (Marinobacter, Pseudomonas, Halomonas and Hydrogenophaga) ensured the rapid removal of nitrogen in the biofilter. When the temperature dropped to 15 degrees C, Pseudomonas progressively took the position of Marinobacter, both of which had denitrification and degradation activities. The relative abundance of the denitrifying bacteria Halomonas that we inoculated has been less than 1 % since phase II. Overall, the PCL-supported CAFB-PADR demonstrated in this study shows potential for removing high concentrations of nitrate from recirculating marine aquaculture wastewater.