Mechanisms of low nighttime temperature promote oil accumulation in Brassica napus L. based on in-depth transcriptome analysis.

Rape (Brassica napus L.; AACC) is an important oil-bearing crop worldwide. Temperature significantly affects the production of oil crops; however, the mechanisms underlying temperature-promoted oil biosynthesis remain largely unknown. In this study, we found that a temperature-sensitive cultivar (O) could accumulate higher seed oil content under low nighttime temperatures (LNT,13°C) compared with a temperature-insensitive cultivar (S). We performed an in-depth transcriptome analysis of seeds from both cultivars grown under different nighttime temperatures. We found that low nighttime temperatures induced significant changes in the transcription patterns in the seeds of both cultivars. In contrast, the expression of genes associated with fatty acid and lipid pathways was higher in the O cultivar than in the S cultivar under low nighttime temperatures. Among these genes, we identified 14 genes associated with oil production, especially BnLPP and ACAA1, which were remarkably upregulated in the O cultivar in response to low nighttime temperatures compared to S. Further, a WGCNA analysis and qRT-PCR verification revealed that these genes were mainly regulated by five transcription factors, WRKY20, MYB86, bHLH144, bHLH95, and NAC12, whose expression was also increased in O compared to S under LNT. These results allowed the elucidation of the probable molecular mechanism of oil accumulation under LNT conditions in the O cultivar. Subsequent biochemical assays verified that BnMYB86 transcriptionally activated BnLPP expression, contributing to oil accumulation. Meanwhile, at LNT, the expression levels of these genes in the O plants were higher than at high nighttime temperatures, DEGs (SUT, PGK, PK, GPDH, ACCase, SAD, KAS II, LACS, FAD2, FAD3, KCS, KAR, ECR, GPAT, LPAAT, PAP, DGAT, STERO) related to lipid biosynthesis were also upregulated, most of which are used in oil accumulation.

Improved YOLOv5 Network for High-Precision Three-Dimensional Positioning and Attitude Measurement of Container Spreaders in Automated Quayside Cranes.

For automated quayside container cranes, accurate measurement of the three-dimensional positioning and attitude of the container spreader is crucial for the safe and efficient transfer of containers. This paper proposes a high-precision measurement method for the spreader's three-dimensional position and rotational angles based on a single vertically mounted fixed-focus visual camera. Firstly, an image preprocessing method is proposed for complex port environments. The improved YOLOv5 network, enhanced with an attention mechanism, increases the detection accuracy of the spreader's keypoints and the container lock holes. Combined with image morphological processing methods, the three-dimensional position and rotational angle changes of the spreader are measured. Compared to traditional detection methods, the single-camera-based method for three-dimensional positioning and attitude measurement of the spreader employed in this paper achieves higher detection accuracy for spreader keypoints and lock holes in experiments and improves the operational speed of single operations in actual tests, making it a feasible measurement approach.

High Spatial and Temporal Resolution NIR-IIb Gastrointestinal Imaging in Mice.

Conventional biomedical imaging modalities, including endoscopy, X-rays, and magnetic resonance, are invasive and insufficient in spatial and temporal resolutions for gastrointestinal (GI) tract imaging to guide prognosis and therapy. Here we report a noninvasive method based on lanthanide-doped nanocrystals with ∼1530 nm fluorescence in the near-infrared-IIb window (NIR-IIb, 1500-1700 nm). The rational design of nanocrystals have led to an absolute quantum yield (QY) up to 48.6%. Further benefiting from the minimized scattering through the NIR-IIb window, we enhanced the spatial resolution to ∼1 mm in GI tract imaging, which is ∼3 times higher compared with the near-infrared-IIa (NIR-IIa, 1000-1500 nm) method. The approach also realized a high temporal resolution of 8 frames per second; thus the moment of mice intestinal peristalsis can be captured. Furthermore, with a light-sheet imaging system, we demonstrated a three-dimensional (3D) imaging on the GI tract. Moreover, we successfully translated these advances to diagnose inflammatory bowel disease.

Upconversion Nonlinear Structured Illumination Microscopy.

Video-rate super-resolution imaging through biological tissue can visualize and track biomolecule interplays and transportations inside cellular organisms. Structured illumination microscopy allows for wide-field super resolution observation of biological samples but is limited by the strong extinction of light by biological tissues, which restricts the imaging depth and degrades its imaging resolution. Here we report a photon upconversion scheme using lanthanide-doped nanoparticles for wide-field super-resolution imaging through the biological transparent window, featured by near-infrared and low-irradiance nonlinear structured illumination. We demonstrate that the 976 nm excitation and 800 nm upconverted emission can mitigate the aberration. We found that the nonlinear response of upconversion emissions from single nanoparticles can effectively generate the required high spatial frequency components in the Fourier domain. These strategies lead to a new modality in microscopy with a resolution below 131 nm, 1/7th of the excitation wavelength, and an imaging rate of 1 Hz.

The effect of manganese exposure on GnRH secretion via Nrf2/mGluR5/COX-2/PGE2/signaling pathway.

There are limited studies focused on the precise mechanism of gonadotropin-releasing hormone (GnRH) secretion dysfunction after overexposure to manganese (Mn). The objective of the present study was to explore the mechanism of Mn disruption of GnRH synthesis via nuclear factor erythroid-2-related factor-2 (Nrf2)/metabotropic glutamate receptor-5 (mGluR5)/cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) signaling pathway in vitro and in vivo. Primary astrocytes were cultured and treated with different doses of Mn, tert-butylhydroquinonet (tBHQ; Nrf2 agonists), 3-[(2-methyl-4-thaizolyl) ethynyl] pyridine (MTEP; mGluR5 inhibitor), and celecoxib (COX-2 inhibitor) to measure the levels of COX-2, mGluR5, Nrf2, and Nrf2 target genes. Mice were randomly divided into 11 groups, of which included the control group, 12.5-, 25-, and 50-mg/kg MnCl2 group, dimethyl sulfoxide (DMSO) group, tBHQ control group, tBHQ pretreatment group, MTEP control group, MTEP pretreatment group, celecoxib control group, and celecoxib pretreatment group. The injection was administered every day for 2 weeks. Then, levels of GnRH, PGE2, COX-2, mGluR5, Nrf2, Nrf2 target genes, and morphological changes in the hypothalamus of mice were measured. Mn reduced protein levels of Nrf2 and mRNA expression of Nrf2 target genes and increased mGluR5, COX-2, PGE2, and GnRH levels. Meanwhile, injury-related histomorphology changes in the hypothalamus of mice were significantly present. In conclusion, excessive exposure to Mn disrupts GnRH secretion through Nrf2/mGluR5/COX-2/PGE2 signaling pathway.

Exonuclease III-Assisted Upconversion Resonance Energy Transfer in a Wash-Free Suspension DNA Assay.

Sensitivity is the key in optical detection of low-abundant analytes, such as circulating RNA or DNA. The enzyme Exonuclease III (Exo III) is a useful tool in this regard; its ability to recycle target DNA molecules results in markedly improved detection sensitivity. Lower limits of detection may be further achieved if the detection background of autofluorescence can be removed. Here we report an ultrasensitive and specific method to quantify trace amounts of DNA analytes in a wash-free suspension assay. In the presence of target DNA, the Exo III recycles the target DNA by selectively digesting the dye-tagged sequence-matched probe DNA strand only, so that the amount of free dye removed from the probe DNA is proportional to the number of target DNAs. Remaining intact probe DNAs are then bound onto upconversion nanoparticles (energy donor), which allows for upconversion luminescence resonance energy transfer (LRET) that can be used to quantify the difference between the free dye and tagged dye (energy acceptor). This scheme simply avoids both autofluorescence under infrared excitation and many tedious washing steps, as the free dye molecules are physically located away from the nanoparticle surface, and as such they remain "dark" in suspension. Compared to alternative approaches requiring enzyme-assisted amplification on the nanoparticle surface, introduction of probe DNAs onto nanoparticles only after DNA hybridization and signal amplification steps effectively avoids steric hindrance. Via this approach, we have achieved a detection limit of 15 pM in LRET assays of human immunodeficiency viral DNA.

[Er3+:Yb3+ co-doped nanocrystals BaGd2ZnO5 of up-conversion optical temperature sensing].

By far, the most efficient upconversion nanocrystals luminescence materials BaGd2ZnO5: 4%Yb3+ , 1%Er3+, with stable chemical performance, were prepared by using Sol-gel method. XRD pattern shows that the sample is pure phase, belongs to the orthogonal crystals, and space group is Pbnm; SEM micrograph shows that the prepared sample of the morphology sized around 150 nm is evenly distributed. Samples with 971 nm semiconductor laser excitation produce a strong green emission, visible to the naked eye, and uponversion strength and pump energy relation n = 1.22 is two-photon for the realization of the upconversion emission. They originated from Er3+ ions 2H(11/2)--2H(11/2)-->4I(15/2) and 4S(3/2)-->4I(15/2) transition emission, Er3+ ions main excited state absorption (ESA) process is: 4I(15/2)-->4I(11/2)-->2F(7/2)-->2H(11/2), 4S(3/2), Yb3+ was added because of its large absorption cross section (10(4) cm(-1)) so that it is easy to transfer excitation energy to the E3+ ions which enhance the layout particles number and the energy state of the 1F7/2, thereby enhancing the intensity of the peaks of the spectrum. Fluorescence intensity ratio (FIR) technique based on the green upconversion emission of the sample has been studied because the Er3+ ions 2H(11/2) and 4S(3/2) energy level spacing is small. The electrons at the two levels conform to the Boltzmann distribution which is a function of temperature, and thus the fluorescence intensity ratio of two levels can be used to measure the temperature of the substrate material. This method does not interfere with temperature field of the measured object, and can eliminate the uncertainty of the accuracy; the test has a wide temperature range and reasonable temperature resolution, the pump source used is simple, convenient and inexpensive, and has more commercial values. The temperature range of the samples is from 350 to 800 K, and the highest temperature measuring sensitivity can reach 0.0031 K(1). At the same time, under low excitation density, it can produce higher conversion transmission power, making it become ideal material for distance non-contact temperature measurement.

Ultraviolet C upconversion fluorescence of trivalent erbium in BaGd2ZnO5 phosphor excited by a visible commercial light-emitting diode.

Multiple ultraviolet (UV) emission bands have been obtained in Er3+ doped BaGd2ZnO5 phosphor under the excitation of a 532 nm solid-state laser, and the emission peaks at 217, 254, 278, 296, 314, 348, 374 and 394 nm were determined to stem from the high-energy states 4D(1/2), 4D(7/2), 2H(9/2), 2P(1/2), 2P(3/2), 4G(7/2), 4G(11/2), 4H(9/2) of trivalent erbium, respectively. Some UV emission bands in the UVC region can be observed when the sample was excited by commercial green (529 nm) and blue (460 nm) LED. In view of the small size, low-drive voltage and price of LED, UVC upconversion phosphor BaGd2ZnO5:Er3+ excited by visible LED has potential application in environmental sciences.

Advancements in manganese complex-based MRI agents: Innovations, design strategies, and future directions.

This review focuses on the advancements in manganese (Mn) complex-based magnetic resonance imaging (MRI) agents for imaging different diseases. Here we emphasize the unique redox properties of Mn to deliver innovative MRI contrast agents, including small molecules, nanoparticles (NPs), metal-organic frameworks (MOFs), and polymer hybrids. Aspects of their rational design have been discussed, including size dependence, morphology tuning, surface property enhancement, etc., while also discussing the existing challenges and potential solutions. The present work will inspire and motivate scientists to emphasize MRI-guided applications and bring clinical success in the coming years.

Breaking barriers in cancer treatment: nanobiohybrids empowered by modified bacteria and vesicles.

Cancer, the leading global cause of mortality, poses a formidable challenge for treatment. The effectiveness of cancer therapies, ranging from chemotherapy to immunotherapy, relies on the precise delivery of therapeutic agents to tumor tissues. Nanobiohybrids, resulting from the fusion of bacteria with nanomaterials, constitute a promising delivery system. Nanobiohybrids offer several advantages, including the ability to target tumors, genetic engineering capabilities, programmed product creation, and the potential for multimodal treatment. Recent advances in targeted tumor treatments have leveraged bacteria-based nanobiohybrids. Here, we outline the progress in cancer treatment using nanobiohybrids. Our focus is particularly on various therapeutic approaches within the context of nanobiohybrid systems, where bacteria are integrated with nanomaterials to combat cancer. It has been demonstrated that bacteria-based nanobiohybrids present a robust and effective method for tumor theranostics.

Effects of Low Nighttime Temperature on Fatty Acid Content in Developing Seeds from Brassica napus L. Based on RNA-Seq and Metabolome.

Brassica napus L. is a vital plant oil resource worldwide. The fatty acid biosynthesis and oil accumulation in its seeds are controlled by several genetic and environmental factors, including daytime and nighttime temperatures. We analyzed changes in oleic and erucic acid content in two double haploid (DH) lines, DH0729, a weakly temperature-sensitive line, and DH0815, a strongly temperature-sensitive line, derived from B. napus plants grown at different altitudes (1600, 1800, 2000, 2200, and 2400 m a.s.l., 28.85° N, 112.35° E) and nighttime temperatures (20/18, 20/16, 20/13 and 20/10 °C, daytime/nighttime temperature). Based on medium- and long-chain fatty acid metabolites, the total oleic acid content 35 and 43 days after flowering was significantly lower in low nighttime temperature (LNT, 20/13 °C) plants than in high nighttime temperature (HNT, 20/18 °C) plants (HNT: 58-62%; LNT: 49-54%; an average decrease of 9%), and the total erucic acid content was significantly lower in HNT than in LNT plants (HNT: 1-2%; LNT: 8-13%; an average increase of 10%). An RNA-seq analysis showed that the expression levels of SAD (LOC106366808), ECR (LOC106396280), KCS (LOC106419344), KAR (LOC106367337), HB1(LOC106430193), and DOF5 (LOC111211868) in STSL seeds increased under LNT conditions. In STSL seeds, a base mutation in the cis-acting element involved in low-temperature responsiveness (LTR), the HB1 and KCS promoter caused loss of sensitivity to low temperatures, whereas that of the KCS promoter caused increased sensitivity to low temperatures.

Transcriptomic Profiling of Shoot Apical Meristem Aberrations in the Multi-Main-Stem Mutant (ms) of Brassica napus L.

Rapeseed (Brassica napus L.) is a globally important oilseed crop with various uses, including the consumption of its succulent stems as a seasonal vegetable, but its uniaxial branching habit limits the stem yield. Therefore, developing a multi-stem rapeseed variety has become increasingly crucial. In this study, a natural mutant of the wild type (ZY511, Zhongyou511) with stable inheritance of the multi-stem trait (ms) was obtained, and it showed abnormal shoot apical meristem (SAM) development and an increased main stem number compared to the WT. Histological and scanning electron microscopy analyses revealed multiple SAMs in the ms mutant, whereas only a single SAM was found in the WT. Transcriptome analyses showed significant alterations in the expression of genes involved in cytokinin (CK) biosynthesis and metabolism pathways in the ms mutant. These findings provide insight into the mechanism of multi-main-stem formation in Brassica napus L. and lay a theoretical foundation for breeding multi-main-stem rapeseed vegetable varieties.

Bone disease imaging through the near-infrared-II window.

Skeletal disorders are commonly diagnosed by X-ray imaging, but the radiation limits its use. Optical imaging through the near-infrared-II window (NIR-II, 1000-1700 nm) can penetrate deep tissues without radiation risk, but the targeting of contrast agent is non-specific. Here, we report that lanthanide-doped nanocrystals can passively target the bone marrow, which can be effective for over two months. We therefore develop the high-resolution NIR-II imaging method for bone disease diagnosis, including the 3D bone imaging instrumentation to show the intravital bone morphology. We demonstrate the monitoring of 1 mm bone defects with spatial resolution comparable to the X-ray imaging result. Moreover, NIR-II imaging can reveal the early onset inflammation as the synovitis in the early stage of rheumatoid arthritis, comparable to micro computed tomography (µCT) in diagnosis of osteoarthritis, including the symptoms of osteophyte and hyperostosis in the knee joint.

NIR-IIb fluorescence-image guided synergistic surgery/starvation/chemodynamic therapy: an innovative treatment paradigm for malignant non-small cell lung cancers.

Background: Currently, the prognosis and survival rate for patients bearing non-small cell lung cancer (NSCLC) is still quite poor, mainly due to lack of efficient theranostic paradigms to exert in time diagnostics and therapeutics. Methods: Herein, for NSCLC treatment, we offer a customized theranostic paradigm, termed NIR-IIb fluorescence diagnosis and synergistic surgery/starvation/chemodynamic therapeutics, with a newly designed theranostic nanoplatform PEG/MnCuDCNPs@GOx. The nanoplatform is composed of brightly NIR-II emissive downconversion nanoparticles (DCNPs)-core and Mn/Cu-silica shell loaded with glucose oxidase (GOx) to achieve synergistic starvation and chemodynamic therapy (CDT). Results: It is found that 10% Ce3+ doped in the core and 100% Yb3+ doped in the middle shell greatly improves the NIR-IIb emission up to even 20.3 times as compared to the core-shell DCNPs without Ce3+ doping and middle shell. The bright NIR-IIb emission of the nanoplatform contributes to sensitive margin delineation of early-stage NSCLC (diameter < 1 mm) with a signal-to-background ratio (SBR) of 2.18, and further assists in visualizing drug distribution and guiding surgery/starvation/chemodynamic therapy. Notably, the starvation therapy mediated by GOx-driven oxidation reaction efficiently depletes intratumoral glucose, and supplies H2O2 to boost the CDT mediated by the Mn2+ and Cu2+, which consequently realized a highly effective synergistic treatment for NSCLC. Conclusion: This research demonstrates an efficient treatment paradigm for NSCLC with NIR-IIb fluorescence diganosis and image-guided synergistic surgery/starvation/chemodynamic therapeutics.

Aspect Ratio of PEGylated Upconversion Nanocrystals Affects the Cellular Uptake In Vitro and In Vivo.

The central nervous system (CNS) is protected by the blood-brain barrier (BBB), which acts as a physical barrier to regulate and prevent the uptake of endogenous metabolites and xenobiotics. However, the BBB prevents most non-lipophilic drugs from reaching the CNS following systematic administration. Therefore, there is considerable interest in identifying drug carriers that can maintain the biostability of therapeutic molecules and target their transport across the BBB. In this regard, upconversion nanoparticles (UCNPs) have become popular as a nanoparticle-based solution to this problem, with the additional benefit that they display unique properties for in vivo visualization. The majority of studies to date have explored basic spherical UCNPs for drug delivery applications. However, the biophysical properties of UCNPs, cell uptake and BBB transport have not been thoroughly investigated. In this study, we described a one-pot seed-mediated approach to precisely control longitudinal growth to produce bright UCNPs with various aspect ratios. We have systematically evaluated the effects of the physical aspect ratios and PEGylation of UCNPs on cellular uptake in different cell lines and an in vivo zebrafish model. We found that PEGylated the original UCNPs can enhance their biostability and cell uptake capacity. We identify an optimal aspect ratio for UCNP uptake into several different types of cultured cells, finding that this is generally in the ratio of 2 (length/width). This data provides a crucial clue for further optimizing UCNPs as a drug carrier to deliver therapeutic agents into the CNS. STATEMENT OF SIGNIFICANCE: The central nervous system (CNS) is protected by the blood-brain barrier (BBB), which acts as a highly selective semipermeable barrier of endothelial cells to regulate and prevent the uptake of toxins and pathogens. However, the BBB prevents most non-lipophilic drugs from reaching the CNS following systematic administration. The proposed research is significant because identifying the aspect ratio of drug carriers that maintains the biostability of therapeutic molecules and targets their transport across the blood-brain barrier (BBB) is crucial for designing an efficient drug delivery system. Therefore, this research provides a vital clue for further optimizing UCNPs as drug carriers to deliver therapeutic molecules into the brain.

QTL mapping for cold tolerance and higher overwintering survival rate in winter rapeseed (Brassica napus).

The cold tolerance of winter rapeseed (Brassica napus) cultivars is critically important for winter survival and yield formation in northern China. Few studies have examined the genetic mechanism underlying the overwintering survival of B. napus. Here, an F2 population including 174 lines and an F2:3 population including 174 lines were generated to identify the quantitative trait loci (QTLs) related to the cold tolerance of B. napus. A genetic linkage map including 1,017 markers merged into 268 bins covering 793.53 cM was constructed. A total of 16 QTLs for two cold-tolerance indicators related to overwintering success were detected among the two populations. These QTLs were responsible for explaining 0.97%-12.74% of the phenotypic variation. Two major QTLs, qOWRTA07 and qOWRLA07, explaining more than 10% of the phenotypic variation were identified in overlapping regions, and we suspected that these two QTLs might represent the same QTL mapped between the two bins, c07b004 and c07b005, corresponding to the physical interval from 21.4 M to 23.4 M on chromosome A07. One gene, BnaA07G0198300ZS, contained the candidate region for overwintering rate (OWR). RT-qPCR analysis showed that the expression of this gene significantly differed between the two parents (NST57 and CY12), and its expression was higher in NST57 than in CY12. This gene may be involved in the cold-response during overwintering period of B. napus. These results are important for the molecular breeding to improve the cold tolerance and overwintering success of winter oilseed rape.

4Kscore diagnostic value in patients with high-grade prostate cancer using cutoff values of 7.5% to 10%: A meta-analysis.

BACKGROUND: Reported 4Kscore thresholds used to differentiate between patients with and without high-grade prostate cancer (CaP) were variable. Patients with 4Kscore results <7.5% have been proven to be at low risk of carrying high-grade CaP. This study employed a meta-analysis approach in order to assess the diagnostic accuracy of the 4Kscore as a means of detecting high-grade CaP in prostate biopsy samples using cutoff values of 7.5% to 10%. METHODS: Relevant studies published as of December 2019 were identified via searching PubMed, Embase, and Cochrane Library. Data pertaining to 4Kscore diagnostic accuracy were then extracted from these studies and utilized for the calculation of pooled sensitivity , specificity , diagnostic odds ratio , and area under the curve values relating to high-grade CaP diagnosis. RESULTS: In total, 9 studies incorporating 1,689 high-grade CaP patients were included in our meta-analysis. Following the exclusion of 1 outlier study, the pooled sensitivity, specificity , diagnostic odds ratio , and area under the curve values for 4Kscore diagnostic accuracy with corresponding 95% confidence intervals (CIs) were 0.90 (95%CI: 0.86-0.92), 0.44 (95%CI: 0.36-0.52), 7 (95%CI: 5-8), and 0.81 (95%CI: 0.77-0.84), respectively. CONCLUSION: These findings indicate that 4Kscore can be used as a model for the diagnosis of high-grade CaP. However, we detected significant heterogeneity among studies that was not explained by subgroup or meta-regression analysis, thus lowering our confidence in these results. It is therefore essential that future large, well-designed studies be conducted so as to confirm whether the 4Kscore can be used with cutoff values of 7.5% to 10% to reliably detect high-grade CaP.

Germinating seed can sense low temperature for the floral transition and vernalization of winter rapeseed (Brassica rapa).

The hybrid production of winter rapeseed is limited by the difficult vernalization processes. Thus, floral regulation of winter rapeseed parental lines cannot be executed through selection of sowing time during hybrid production. Therefore, in this study, strong winter rapeseed was used as the material to analyse the floral transition mechanism of germinating seed vernalization. Results demonstrated that germinating seeds could sense low temperatures and complete vernalization following a low temperature treatment for 56.5 d with a 100 % vernalization rate. The regression equation between vernalization rate (y) and vernalization treatment days (x) was determined as y = 0.019x - 0.0765 (R² = 0.8529). When the vernalization treatment time was prolonged, the vernalization rate and fruiting ability increased rapidly, and variations were observed in the membrane lipid oxidation and physiological characteristics. Furthermore, at the prolonged treatment time of 10-50 d, the salicylic acid (SA) content continued to decrease, with values significantly lower than those of the control. SA content is significantly positively correlated with the level of BrFLC transcription and a significantly negatively correlated with the vernalization rate of germinating seeds. Moreover, the expressions of genes associated with SA biosynthesis, SA signal transduction, the flowering key negative regulators were suppressed and that of positive regulators were promoted during vernalization. These results suggest that SA as a floral repressor is involved in the regulation of the vernalization process of winter rapeseed germination seeds. In addition, SA may be related to the counting dosage of vernalization.

Comparative proteomics analysis reveals the molecular mechanism of enhanced cold tolerance through ROS scavenging in winter rapeseed (Brassica napus L.).

Two winter rapeseed cultivars, "NS" (cold tolerant) and "NF" (cold sensitive), were used to reveal the morphological, physiological, and proteomic characteristics in leaves of plants after treatment at -4°C for 12 h(T1) and 24 h(T2), and at room temperature(T0), to understand the molecular mechanisms of cold tolerance. Antioxidant activity and osmotic adjustment ability were higher, and plasma membrane injury was less obvious, in NS than in NF under cold stress. We detected different abundant proteins (DAPs) related to cold tolerance in winter rapeseed through data-independent acquisition (DIA). Compared with NF, A total of 1,235 and 1,543 DAPs were identified in the NSs under T1 and T2, respectively. Compared with NF, 911 proteins were more abundant in NS only after cold treatment. Some of these proteins were related to ROS scavenging through four metabolic pathways: lysine degradation; phenylalanine, tyrosine, and tryptophan; flavonoid biosynthesis; and ubiquinone and other terpenoid-quinone biosynthesis. Analysis of these proteins in the four candidate pathways revealed that they were rapidly accumulated to quickly enhance ROS scavenging and improve the cold tolerance of NS. These proteins were noticeably more abundant during the early stage of cold stress, which was critical for avoiding ROS damage.

Overexpression of BrAFP1 gene from winter rapeseed (Brassica rapa) confers cold tolerance in Arabidopsis.

Antifreeze proteins (AFPs) can bind to ice crystals and restrain the formation of larger crystals, a strategy vital to the survival of plants in freezing environments. The BrAFP1 from winter rapeseed cultivars 'Longyou 7' with high cold tolerance was cloned and overexpressed in Arabidopsis. BrAFP1 was localized in the cytoplasm and nucleus. Under cold stress, SOD activity and free proline content were higher, MDA content and relative conductivity were lower in transgenic lines than those in wide-type Arabidopsis. Frostbite of transgenic plants was minimized, whereas frostbite of the Arabidopsis afp1 mutant was severe. Transition of the amino acid at position 17 of BrAFP1 was related to the increased winter survival of the rapeseed cultivar. Cultivars with higher survival rates had a predilection for tyrosine, not tryptophan, at the 17th site in the amino sequence of BrAFP1. Transcription of BrAFP1 was induced more rapidly, and the expression of the gene was also higher, in Longyou 7 than that in Tianyou 4 under cold stress. Overall, the high expression of BrAPF1 confers more cold-tolerance in Longyou 7.