Leaf color formation mechanisms in Alternanthera bettzickiana elucidated by metabolite and transcriptome analyses.

MAIN CONCLUSION: The difference in leaf color among the three cultivars of A. bettzickiana is due to different chloroplast morphology and chlorophyll-to-anthocyanin ratios. Alternanthera bettzickiana is one of the most important ornamental plants in modern flower beds because of its colorful leaves. The present study examined the mechanism of leaf color formation in A. bettzickiana. Three cultivars of A. bettzickiana (red, green, and mixed red and green) were selected for comprehensive analyses of leaf color formation by examining cellular and subcellular structures and pigment biosynthesis and metabolism. The difference in leaf colors between the three cultivars of A. bettzickiana was due to different chlorophyll-to-anthocyanin ratios. A. bettzickiana 'Green' showed very low expression of CHS, F3H, and DFR, the key genes of the anthocyanin biosynthesis pathway, and a low anthocyanin content but had mature chloroplasts and a green color. A. bettzickiana 'Red' exhibited a low chlorophyll content and deformed chloroplasts but a high cyanidin content and, thus, a red color. A. bettzickiana 'Variegated' presented high anthocyanin and chlorophyll contents and exhibited red and green variegation, indicating a balance between coloration and photosynthetic efficiency. These data provide a good explanation for the coloration of different cultivars of A. bettzickiana and an important reference for better explaining the color formation mechanisms of plant leaves.

The nonreciprocal heterostyly and heterotypic self-incompatibility of Ceratostigma willmottianum.

Ceratostigma willmottianum (Plumbaginaceae) is a perennial herb native to China. Many species of Plumbaginaceae have been reported to exhibit heterostyly. Determining the functional breeding system of C. willmottianum can improve our understanding of the reproductive ecology of heterostylous plants. We investigated the floral traits and pollen and stigma characteristics in a natural population, and artificial pollination was carried out in an artificial population. It was found that C. willmottianum was distylous with short (S)- and long (L)-styled morphs, did not exhibit precise reciprocal herkogamy and was partially self-compatible but primarily outcrossing. In the artificial pollination experiments, the pollen tubes reached the base of the style under intermorph pollination, whereas they rarely penetrated the style under intramorph pollination and self-pollination. Both the L and S morphs exhibited a high seed set after intermorph pollination, whereas the seed set under intramorph pollination was lower. Therefore, C. willmottianum may spread the chance of receiving pollen between the two morphs by nonreciprocal heterostyly, which may be a unique mode of ecological adaptation in Plumbaginaceae. We believe that our discovery could provide new ideas regarding the origin and evolution of heterostyly.

Exploration of the Potential Transcriptional Regulatory Mechanisms of DNA Methyltransferases and MBD Genes in Petunia Anther Development and Multi-Stress Responses.

Cytosine-5 DNA methyltransferases (C5-MTases) and methyl-CpG-binding-domain (MBD) genes can be co-expressed. They directly control target gene expression by enhancing their DNA methylation levels in humans; however, the presence of this kind of cooperative relationship in plants has not been determined. A popular garden plant worldwide, petunia (Petunia hybrida) is also a model plant in molecular biology. In this study, 9 PhC5-MTase and 11 PhMBD proteins were identified in petunia, and they were categorized into four and six subgroups, respectively, on the basis of phylogenetic analyses. An expression correlation analysis was performed to explore the co-expression relationships between PhC5-MTases and PhMBDs using RNA-seq data, and 11 PhC5-MTase/PhMBD pairs preferentially expressed in anthers were identified as having the most significant correlations (Pearson's correlation coefficients > 0.9). Remarkably, the stability levels of the PhC5-MTase and PhMBD pairs significantly decreased in different tissues and organs compared with that in anthers, and most of the selected PhC5-MTases and PhMBDs responded to the abiotic and hormonal stresses. However, highly correlated expression relationships between most pairs were not observed under different stress conditions, indicating that anther developmental processes are preferentially influenced by the co-expression of PhC5-MTases and PhMBDs. Interestingly, the nuclear localization genes PhDRM2 and PhMBD2 still had higher correlations under GA treatment conditions, implying that they play important roles in the GA-mediated development of petunia. Collectively, our study suggests a regulatory role for DNA methylation by C5-MTase and MBD genes in petunia anther maturation processes and multi-stress responses, and it provides a framework for the functional characterization of C5-MTases and MBDs in the future.

Fipronil and its metabolites in human seminal plasma from Shijiazhuang, north China.

Fipronil (FP) is an emerging insecticide which could induce reproductive toxicity in male rats at very low dosage, but the occurrence of FP and its transformation products (FPs) in human seminal plasma and their impacts on human semen quality have not been documented. In this study, FPs including FP, fipronil desulfinyl (FP-DES), fipronil sulfone (FP-SFO), fipronil amide (FP-AM), and fipronil sulfide (FP-SFI), were measured in seminal plasma samples (n = 200), which were collected from Shijiazhuang, north China. The cumulative concentration of FPs (ΣFPs), in the seminal plasma samples ranged from 0.003 to 0.180 ng/mL (median: 0.043 ng/mL). FP-SFO was the major target analyte (median: 0.040 ng/mL), accounting for approximately 42.3-100.0% of the ΣFPs. Significantly higher exposure levels of FPs were found in the overweight or obese group (≥25 kg/m2) vs. the normal BMI group (18.5-25 kg/m2) (ΣFPs: 0.047 vs. 0.033 ng/mL), never smoking group vs. current smoking group (ΣFPs: 0.057 vs. 0.037 ng/mL), and low sexual frequency group (<1 time/week) vs. high sexual frequency group (≥3 times/week) (ΣFPs: 0.048 vs. 0.030 ng/mL). No significant association between FPs and impaired semen quality parameter was found in this study. This is the first time to report FPs' occurrence in human seminal plasma and variations in their concentrations among people with different demographic and behavioral characteristics. Further studies on adverse effects of exposure to FPs on reproductive function are needed.

Transcriptome Analysis Revealed a Cold Stress-Responsive Transcription Factor, PaDREB1A, in Plumbago auriculata That Can Confer Cold Tolerance in Transgenic Arabidopsis thaliana.

The tropical plant Plumbago auriculata can tolerate subzero temperatures without induction of apoptosis after cold acclimation in autumn, making it more cold tolerant than conventional tropical plants. In this study, we found that low temperatures significantly affected the photosynthetic system of P. auriculata. Using transcriptome sequencing, PaDREB1A was identified as a key transcription factor involved in the response to cold stress in P. auriculata. This transcription factor may be regulated by upstream JA signaling and regulates downstream ERD4 and ERD7 expression to resist cold stress. Overexpression of PaDREB1A significantly enhanced freezing resistance, protected the photosynthetic system, and enhanced the ROS scavenging mechanism under cold stress in Arabidopsis thaliana. Additionally, PaDREB1A significantly enhanced the expression of CORs and CAT1 in A. thaliana, which further activated the downstream pathway to enhance plant cold tolerance. This study explored the possible different regulatory modes of CBFs in tropical plants and can serve as an important reference for the introduction of tropical plants to low-temperature regions.

Insecticide fipronil and its transformation products in human blood and urine: Assessment of human exposure in general population of China.

Fipronil (FP) is widely used as a highly effective insecticide worldwide, thereby raising concern about environmental contamination and risk for human health. However, data on the occurrence of FP and its transformation products (FPs) in human blood and urine are limited. In this study, 39 pairs of serum, plasma, blood cells (BCs), and urine samples were collected from adults in Wuhan, central China (2020), in order to characterize the concentration profiles of FPs in different matrices. FPs were also determined in serum samples (n = 226, including 57, 56, 56, and 57 samples for Wuhan, Huangshi, Nanjing, and Zhenjiang, respectively) collected from four cities of China (2015) to characterize the exposure levels of FPs among the general population and potential spatial variations. Fipronil sulfone (FP-SFO) was 100% detectable in blood samples, and it was the predominant metabolite (accounting for 86-95% of the cumulative concentrations of FPs [ΣFPs]), with the median concentrations (ng/mL) of 0.17, 0.16, and 0.03 in serum (range: 0.07-1.53), plasma (range: 0.06-1.41), and BCs (range: 0.01-0.24), respectively. The compositional profiles of FPs in serum, plasma, and BCs were similar; very strong positive correlations were observed between different blood matrices for FP-SFO (r = 0.94-0.97, p < 0.01) but not between blood and urine. The median ΣFPs (ng/mL) in the serum (0.20; range: 0.09-1.56) and the plasma samples (0.19; range: 0.09-1.43) was higher than that in BCs samples (0.04; range: 0.01-0.24). In the urine samples, only the major metabolite FP-SFO was detectable in approximately 10% of the samples. Additionally, the highest median ΣFPs (ng/mL) in the serum samples was found in Nanjing (0.56; range: 0.13-1.88), followed by Wuhan (0.34; range: 0.06-1.02), Huangshi (0.10; range: 0.03-0.60), and Zhenjiang (0.08; range: 0.02-0.42). The level of ΣFPs seemed to increase with city sizes and urbanization scale, though further studies are needed to confirm the variations with larger sample size. The estimated daily intake of ΣFPs based on the median concentration of samples from Nanjing (18.5 ng/kg-bw/d) was higher than that of Wuhan (11.3), Huangshi (3.40), and Zhenjiang (2.80). Dietary intake should be the major exposure route for the general population, while water or indoor dust accounted for <1% of the ΣFPs intake. This pilot study provided the first data on the profiles of FPs in paired human serum, plasma, BCs, and urine samples, and potential spatial variations of ΣFPs in China. FP-SFO and FP desulfinyl should be considered among priority substances worthy to be bio-monitored in China due to its moderated persistence and ubiquitous occurrence in human blood.

Selected transformation products of neonicotinoid insecticides (other than imidacloprid) in drinking water.

Several transformation products (or metabolites) of neonicotinoid insecticides (NNIs) have been detected in drinking water, such as desnitro-imidacloprid and imidacloprid-urea. However, data on the occurrences of the metabolites of NNIs (mNNIs) in drinking water are mainly limited to the imidacloprid metabolites. To identify whether the potential metabolites of other widely used NNIs (such as acetamiprid, clothianidin, and thiamethoxam) occur in drinking water and to characterize their distribution profiles, twelve selected (mainly urea and desnitro/decyano) metabolites of NNIs were measured in drinking water samples (n = 884, including n = 789 for tap water, and n = 95 for shallow groundwater) that were collected from 32 provinces in mainland China and Hong Kong. Nearly 90% of the drinking water samples contained the detected mNNI residues. Among the selected mNNIs, thiamethoxam-urea (THM-urea: 76%) and decyano-acetamiprid (decyano-ACE: 73%) were frequently detected (median: 0.94 and 0.25 ng/L, respectively), which were followed by clothianidin-urea (CLO-urea: 45%), desnitro-thiamethoxam (DN-THM: 38%), and other mNNIs (detected in less than 30% of the water samples). Surface-water-sourced tap water had an approximately 8-10 times higher median cumulative concentration (ng/L) of the selected mNNIs (ΣmNNIs: 3.88) than the deep groundwater-sourced tap water (0.53) and groundwater that was directly used as drinking water (0.38). Higher ratios of THM-urea accounted for ΣTHM in north and northwest China than in south China could be partly explained by the decreasing soil pH values from north to south in China. The higher ratios of decyano-ACE accounted for ΣACE in south China than in north and northwest China could be attributable to the lower soil pH levels, higher temperatures, and greater light intensities in south China. The THM-urea, decyano-ACE, and ΣmNNIs levels in cities were found to be significantly higher than those in nonurban areas. The THM-urea levels in seven drinking water samples from Guangxi and Henan Provinces exceeded the guideline limit (100 ng/L) of the European Union. This is the first study to identify THM-urea, decyano-ACE, CLO-urea, and DN-THM in drinking water. To better assess the mass loadings of NNIs in drinking water, mNNIs should be considered in further studies.

A nationwide study of occurrence and exposure assessment of neonicotinoid insecticides and their metabolites in drinking water of China.

Neonicotinoid insecticides (NNIs) are the most extensively used insecticides worldwide, threatening ecosystem and human health. However, nationwide studies of NNIs and their metabolites in drinking water are limited. In order to characterize the contamination status of NNIs in drinking water throughout China, we collected 884 drinking water samples including 789 tap water and 95 groundwater samples from 32 provinces (covering seven regions of mainland China: south, central, east, north, northeast, northwest, and southwest) and Hong Kong. Ten NNIs and six of their main metabolites were determined in the water samples. The relative potency factor method was used to assess the cumulative concentrations of NNIs and their metabolites (imidacloprid-equivalent total NNIs, IMIeq) based on the chronic reference doses (cRfDs) of the NNIs or the toxic effects of the mataboilites. The IMIeq varied among the studied regions, with a median concentration of 24.5 ng/L and a maximum concentration of 8,622 ng/L. The predominant NNIs in drinking water were acetamiprid (ACE) and imidacloprid (IMI). Compared with tap water derived from groundwater, much higher concentrations of IMIeq and NNIs were found in tap water derived from surface water. Different concentrations and patterns of NNIs in drinking water were observed in different regions, provinces, and capital cities, mainly due to regional and provincial differences in crop types and volumes of pesticide usage. The concentrations of NNIs in the drinking water of provincial capitals and small/medium cities were higher than the concentrations in rural areas. The estimated daily intake (EDI) of IMIeq was at least two orders of magnitude lower than the cRfD of IMI, while the NNIs in 16 drinking water samples exceeded the acceptable value (100 ng/L) recommended by the European Union. This study provided a nationwide profile of the occurrence of NNIs and their metabolites in the drinking water of China and the associated potential cumulative human health risks, taking into account of the toxicity differences between NNIs and their metabolites.

Spatial variation of fipronil and its derivatives in tap water and ground water from China and the fate of them during drinking water treatment in Wuhan, central China.

Fipronil (FP) is an emerging insecticide, however, its occurrence in drinking water and source water is limited. In this work, a total of 789 tap water and 95 ground water samples were collected from China in June 2019 in order to assess occurrence of FP and its derivatives (FPs). FPs were also analyzed in source, treated (n = 10, July), and tap water samples (n = 81, July and October 2019) originating from the central Yangtze River and its tributary, the Hanshui River in Wuhan. The sum concentrations of FPs (ΣFPs) in the tap water in China ranged from not detected (ND) to 5.07 (median: 0.03 ng/L), with FP found in 55.3% of the samples, and other targets ≤ 50.0%. Significant regional variations in the ΣFPs values were found between East China (75th percentile: 0.31 ng/L) and Northwest China (0.04), as well as between East China and North China (0.04). Similar ΣFPs values were found for ground water and tap water. The estimated daily intake of ΣFPs via water ingestion was below 200 pg/kg-bw/day for all age groups and was lower than the reference dose for FP (0.2 µg/kg-bw/day). Additionally, FPs were found in all of the source water samples collected in Wuhan with concentrations in the range of 0.84-2.72 ng/L for ΣFPs (median: 2.39). Most of these FPs were removed during water treatment. Higher concentration of ΣFPs in tap water was observed in July (median: 0.04 ng/L) compared to that in October (ND). This is the first study on the occurrence of FPs in the Yangtze River, the fate of FPs during the tap water treatment, and the regional distribution of FPs in tap water from China.

Occurrence of the insecticide fipronil and its degradates in indoor dust from South, Central, and North China.

No known data are available regarding the occurrence of fipronil (FP) in indoor dust in China, despite the fact that it is an emerging insecticide that is used extensively. In this study, FP and its derivatives (FPs), including fipronil sulfone (FP-SFO), fipronil sulfide, fipronil desulfinyl, and fipronil amide, were determined in indoor dust samples (n = 436) collected from three different cities in China between 2016 and 2019, namely Shenzhen (February and September 2019), Wuhan (October 2016, October 2018, and August 2019), and Taiyuan (October 2016), located in south, central, and north China, respectively. FP and FP-SFO were the major target analytes detected in all dust samples, accounting for approximately 68.6-90.1% of the cumulative concentration of FPs (ΣFPs). The ΣFPs values for the dust samples ranged from not detected (ND) to 33.6 µg/g (median ± median absolute deviation [MAD]: 7.12 ± 6.44 ng/g), with a detection frequency of 95.6%. The median value of the ΣFPs of the dust samples collected from Shenzhen (22.6 ± 15.6 ng/g) was higher than that found in the dust samples collected in Taiyuan 2016 (1.87 ± 1.65 ng/g) and Wuhan (6.43 ± 5.62 ng/g). Significantly higher ΣFPs were observed in the dust samples collected in urban areas than in the rural areas of Taiyuan and Wuhan. Furthermore, an increasing trend of FP (from 2016 to 2018) in the rural areas of Wuhan, and a seasonal variation (summer > autumn) in FP, FP-SFO, and ΣFPs in the dust samples from urban areas of Wuhan were observed. The estimated daily intake of FPs via dust ingestion for people in Shenzhen was approximately 12 times higher than in Taiyuan based on the samples analyzed. This is the first study to describe the occurrence and spatiotemporal variations of FPs in indoor dust in China.