Detection and specific chemical identification of submillimeter plastic fragments in complex matrices such as compost.

Research on the plastic contamination of organic fertilizer (compost) has largely concentrated on particles and fragments > 1 mm. Small, submillimeter microplastic particles may be more hazardous to the environment. However, research on their presence in composts has been impeded by the difficulty to univocally identify small plastic particles in such complex matrices. Here a method is proposed for the analysis of particles between 0.01 and 1.0 mm according to number, size, and polymer type in compost. As a first demonstration of its potential, the method is used to determine large and small microplastic in composts from eight municipal compost producing plants: three simple biowaste composters, four plants processing greenery and cuttings and one two-stage biowaste digester-composter. While polyethylene, PE, tends to dominate among fragments > 1 mm, the microplastic fraction contained more polypropylene, PP. Whereas the contamination with PE/PP microplastic was similar over the investigated composts, only composts prepared from biowaste contained microplastic with a signature of biodegradable plastic, namely poly(butylene adipate co-terephthalate), PBAT. Moreover, in these composts PBAT microplastic tended to form the largest fraction. When the bulk of residual PBAT in the composts was analyzed by chloroform extraction, an inverse correlation between the number of particles > 0.01 mm and the total extracted amount was seen, arguing for breakdown into smaller particles, but not necessarily a mass reduction. PBAT oligomers and monomers as possible substrates for subsequent biodegradation were not found. Remaining microplastic will enter the environment with the composts, where its subsequent degradability depends on the local conditions and is to date largely uninvestigated.

Role of Residual Monomers in the Manifestation of (Cyto)toxicity by Polystyrene Microplastic Model Particles.

Polystyrene (PS) is an important model polymer for the investigation of effects of microplastic (MP) and nanoplastic (NP) particles on living systems. Aqueous dispersions of PS MP or NP contain residual monomers of styrene. In consequence, it is not clear if the effects observed in standard (cyto)toxicity studies are evoked by the polymer (MP/NP) particle or by residual monomers. We addressed that question by comparing standard PS model particle dispersions with in-house synthesized PS particle dispersions. We proposed a rapid purification method of PS particle dispersions by dialysis against mixed solvents and developed a simple method of UV-vis spectrometry to detect residual styrene in the dispersions. We found that standard PS model particle dispersions, which contain residual monomers, exerted a low but significant cytotoxicity on mammalian cells, while the in-house synthesized PS, after rigorous purification to reduce the styrene content, did not. However, the PS particles per se but not the residual styrene in both PS particle dispersions resulted in immobilization of Daphnia. Only by using freshly monomer-depleted particles, will it be possible in the future to assess the (cyto)toxicities of PS particles, avoiding an otherwise not controllable bias effect of the monomer.

Overexpression of major latex protein 423 (NtMLP423) enhances the chilling stress tolerance in Nicotiana tabacum.

Chilling stress impedes plant growth and hinders crop development and productivity. In this study, we identified the major latex protein (MLP) in tobacco (NtMLP423) and examined its roles in chilling resistance. NtMLP423 expression was considerably upregulated in response to chilling stress. NtMLP423 function was assessed and compared in plants with overexpression and antisense characteristics. Under chilling stress, plants with overexpression characteristics grew better than wild-type and antisense plants. NtMLP423 overexpression reduced membrane lipid damage, increased antioxidant enzyme activity, and reduced reactive oxygen species (ROS) accumulation under chilling stress. Here, we screened for the first time the upstream transcription factor NtMYB108, which regulates NtMLP423 expression under chilling stress. The NtMYB108 transcription factor directly binds to the NtMLP423 promoter and improves NtMLP423 resistance to chilling stress. Subjecting NtMYB018 to virus-induced gene silencing reduced chilling stress tolerance. Overall, NtMLP423 overexpression enhances chilling stress tolerance, while its suppression has the opposite effect.

Over-Expression of an R2R3 MYB Gene, MdMYB108L, Enhances Tolerance to Salt Stress in Transgenic Plants.

Plants are affected by various abiotic stresses during their growth and development. In plants, MYB transcription factors are involved in various physiological and biochemical processes, including biotic and abiotic stress responses. In this study, we functionally analyzed MdMYB108L. We examined the transcriptional activity of MdMYB108L under salt stress and determined that the N-terminal domain of MdMYB108L, which was significantly induced under salt stress, has transcriptional activity. MdMYB108L overexpression increased the germination rate, main root length, and the antioxidant activity of catalase and peroxidase in transgenic Arabidopsisthaliana seeds, while reducing reactive oxygen species (ROS) accumulation. MdMYB108L overexpression also increased the photosynthetic capacity of hairy root tissue (leaves) under salt stress. In addition, the MdMYB108L transcription factor bound to the MdNHX1 promoter positively regulated the transcription of the salt tolerance gene MdNHX1 in apples, improving the salt stress tolerance of transgenic plants. These findings have implications for improving the agricultural yields of apple trees under salt stress.

Municipal biowaste treatment plants contribute to the contamination of the environment with residues of biodegradable plastics with putative higher persistence potential.

Biodegradable plastics (BDP) are expected to mineralize easily, in particular under conditions of technical composting. However, the complexity of the sample matrix has largely prevented degradation studies under realistic conditions. Here composts and fertilizers from state-of-the-art municipal combined anaerobic/aerobic biowaste treatment plants were investigated for residues of BDP. We found BDP fragments > 1 mm in significant numbers in the final composts intended as fertilizer for agriculture and gardening. Compared to pristine compostable bags, the recovered BDP fragments showed differences in their material properties, which potentially renders them less prone to further biodegradation. BDP fragments < 1 mm were extracted in bulk and came up to 0.43 wt% of compost dry weight. Finally, the liquid fertilizer produced during the anaerobic treatment contained several thousand BDP fragments < 500 µm per liter. Hence, our study questions, if currently available BDP are compatible with applications in areas of environmental relevance, such as fertilizer production.

Metabolome and transcriptome profiling provide insights into green apple peel reveals light- and UV-B-responsive pathway in anthocyanins accumulation.

BACKGROUND: In nature, green apple are associated with the accumulation of chlorophyll, while red apple varieties are associated with anthocyanins accumulation. Notably, in this study, the green skin color apple variety 'white winter pearmain' treated with ultraviolet-B (UV-B) exhibited red skins and marked anthocyanin accumulation, while visible light could not. But there are few reports on the biosynthesis difference of anthocyanins in green apple by visible light and UV-B-treatment. Here, we explored the difference of metabolites and genes expression level in green apple by transcriptomic and metabolic. RESULTS: The metabolic analysis revealed that there were 152 and 178 significantly changed metabolites in the visible light and UV-B-treated green apple, respectively, compared to the control, and flavone, flavonol, and anthocyanin were the most significantly increased; and transcriptomic analysis showed that 37,110 and 37,709 differentially expressed genes, including 382 and 475 transcription factors (TFs) were detected in light and UV-B-treatment fruit, respectively. Quantitative reverse transcription PCR (qRT-PCR) results confirmed changes in the expression levels of genes encoding metabolites involved in the flavonoid synthesis pathways. The flavonoid metabolic flux in the UV-B treatment increased the accumulation of cyanidin 3-glucoside and cyanidin 3, 5-diglucoside compared to under the light-treatment. Furthermore, we performed qRT-PCR analysis of anthocyanin biosynthesis genes and predicted the gene of MD00G1134400 (a UDP glucose-flavonoid 3-0-glucosyltransferase) may be a candidate gene for anthocyanins accumulation and highly expressed in UV-B-treatment fruit. Expression profiles of several transcription factors of the families MYB, bHLH, NAC were highly correlated with the content of the anthocyanin. CONCLUSIONS: The composition and contents of anthocyanins in green apple in UV-B-treatment very greatly. A series of metabolites and candidate genes were revealed through combined analysis of metabolome and transcriptome. These results provide an important data for dissecting candidate genes and molecular basis governing green apple color formation in response to visible light and UV-B light.

De novo transcriptome assembly of transgenic tobacco (Nicotiana tabacum NC89) with early senescence characteristic.

The enzyme, α-farnesene synthase (AFS), which synthesizes α-farnesene, is the final enzyme in α-farnesene synthesis pathway. We overexpressed the α-farnesene synthase gene (previously cloned in our lab from apple peel) and ectopically expressed it in tobacco (Nicotiana tabacum NC89). Then, the transgenic plants showed an accelerated developmental process and bloomed about 7 weeks earlier than the control plants. We anticipate that de novo transcriptomic analyses of N. tabacum may provide useful information on isoprenoid biosynthesis, growth, and development. We generated 318,925,338 bp sequencing data using Illumina paired-end sequencing from the cDNA library of the apical buds of transgenic line and the wild-type line. We annotated and functionally classified the unigenes in a nucleotide and protein database. Differentially expressed unigenes may be involved in carbohydrate metabolism, nitrogen metabolism, transporter activity, hormone signal transduction, antioxidant systems and transcription regulator activity particularly related to senescence. Moreover, we analyzed eight genes related to terpenoid biosynthesis using qRT-PCR to study the changes in growth and development patterns in the transgenic plants. Our study shows that transgenic plants show premature senescence. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00953-z.

Aloe vera gel coating aggravates superficial scald incidence in 'Starking' apples during low-temperature storage.

The effects of aloe vera (Aloe vera (L.) Burm. f.) gel treatment on the incidence of superficial scald in 'Starking' apples (Malus domestica Borkh. Var. Starking) during cold storage were studied. Apples were harvested at the pre-climacteric stage and treated with aloe vera gel. The treatment increased malondialdehyde content and membrane lipid damage. Furthermore, it inhibited the release of ethylene at the early stage but increased it in the later stage. The expression level of ACC synthase 1 (MdACS1) also increased, and the antioxidant capacity in apples, particularly, catalase, peroxidase, and superoxide dismutase activities, all decreased, while concomitantly, the content of α-farnesene and its oxidation product, conjugated triene increased, thereby aggravating superficial scald incidence during storage at low temperature.

The Nicotiana tabacum L. major latex protein-like protein 423 (NtMLP423) positively regulates drought tolerance by ABA-dependent pathway.

BACKGROUND: Drought stress is an environmental factor that limits plant growth and reproduction. Little research has been conducted to investigate the MLP gene in tobacco. Here, NtMLP423 was isolated and identified, and its role in drought stress was studied. RESULTS: Overexpression of NtMLP423 improved tolerance to drought stress in tobacco, as determined by physiological analyses of water loss efficiency, reactive oxygen species levels, malondialdehyde content, and levels of osmotic regulatory substances. Overexpression of NtMLP423 in transgenic plants led to greater sensitivity to abscisic acid (ABA)-mediated seed germination and ABA-induced stomatal closure. NtMLP423 also regulated drought tolerance by increasing the levels of ABA under conditions of drought stress. Our study showed that the transcription level of ABA synthetic genes also increased. Overexpression of NtMLP423 reduced membrane damage and ROS accumulation and increased the expression of stress-related genes under drought stress. We also found that NtWRKY71 regulated the transcription of NtMLP423 to improve drought tolerance. CONCLUSIONS: Our results indicated that NtMLP423-overexpressing increased drought tolerance in tobacco via the ABA pathway.

MdMYC2 and MdERF3 Positively Co-Regulate α-Farnesene Biosynthesis in Apple.

α-Farnesene, a sesquiterpene volatile compound plays an important role in plant defense and is known to be associated with insect attraction and with superficial scald of apple and pear fruits during cold storage. But the mechanism whereby transcription factors regulate apple α-farnesene biosynthesis has not been clarified. Here, we report that two transcription factors, MdMYC2 and MdERF3 regulated α-farnesene biosynthesis in apple fruit. Dual-luciferase assays and Y1H assays indicated that MdMYC2 and MdERF3 effectively trans-activated the MdAFS promoter. EMSAs showed that MdERF3 directly binds the DRE motif in the MdAFS promoter. Subsequently, overexpression of MdMYC2 and MdERF3 in apple calli markedly activated the transcript levels of MdHMGR2 and MdAFS. Furthermore, transient overexpression of MdMYC2 and MdERF3 in apple fruit significantly increased MdAFS expression and hence, α-farnesene production. These results indicate that MdMYC2 and MdERF3 are positive regulators of α-farnesene biosynthesis and have important value in genetic engineering of α-farnesene production.

The 3-hydroxy-3-methylglutaryl-coenzyme A reductase 5 gene from Malus domestica enhances oxidative stress tolerance in Arabidopsis thaliana.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is the first rate-limiting enzyme regulating the synthesis of terpenoids upstream of the mevalonate (MVA) pathway. In higher plants, members of the HMGR genes families play an important role in plant growth and development and in response to various environmental stresses. In the present study, a novel HMGR gene, designated MdHMGR5, was isolated from apple (Malus domestica L.) and characterized. Expression of MdHMGR5 enhanced the activity of HMGR enzyme in transgenic Arabidopsis thaliana L. plants. Under oxidative stress, transgenic A. thaliana plants over-expressing MdHMGR5 had a higher germination rate, a longer main root length, higher chlorophyll and proline content, and higher activities of antioxidant enzymes. On the other hand, malondialdehyde (MDA) content, relative conductivity and reactive oxygen species (ROS) production rate were significantly lower than in wild type plants. These results indicated that over-expression of MdHMGR5 enhanced plant tolerance to oxidative stress by scavenging ROS in transgenic plants. Over-expression of MdHMGR5 also affected the expression levels of genes in mevalonic acid and 2C-methyl-D-erythritol 4-phosphate (MVA and MEP) pathways of A. thaliana plants. These results indicate that over-expression of MdHMGR5 enhances tolerance to oxidative stress by maintaining photosynthesis and scavenging ROS in transgenic A. thaliana plants.

Transcriptomic profiling revealed genes involved in response to cold stress in maize.

Maize is an important food crop. Chilling stress can decrease maize production by affecting seed germination and seedling growth, especially in early spring. We analysed chlorophyll fluorescence, membrane lipids, secondary metabolites and the transcriptome of two maize inbred lines (chilling-tolerant M54 and chilling-sensitive 753F) after 0, 4 and 24 h cold stress. M54 showed better ability to protect PSII and accumulate secondary metabolites. From RNA sequencing data, we determined that the majority of cold-affected genes were involved in photosynthesis, secondary metabolism, and signal transduction. Genes important for maintaining photosystem structure and for regulating electron transport were less affected by cold stress in M54 than in 753F. Expression of genes related to secondary metabolism and unsaturated fatty acid synthesis were upregulated more strongly in M54 than in 753F and M54 accumulated more unsaturated fatty acids and secondary metabolites. As a result, M54 achieved relatively high cold tolerance by protecting the photosystems and maintaining the stability of cell membranes.

Genome-wide analysis of zinc- and iron-regulated transporter-like protein family members in apple and functional validation of ZIP10.

Deficiency of zinc (Zn) and iron (Fe) is common in apple grown in orchards, which affects fruit yield and quality. However, the mechanisms of absorption and transport of Zn and Fe in apples are still unclear. In the present study, we aimed to identify MdZIP genes and explore the mechanism of response of MdZIPs to Zn and Fe deficiencies. Eighteen Zn- and Fe-regulated transporter-like protein (ZIP) family members were identified in apple (Malus domestica L.) and named according to their chromosomal location. Phylogenetic analysis divided MdZIPs into four groups, and the most closely related MdZIPs in the phylogenetic tree showed similar gene structures and protein motifs. Expression pattern analysis indicated that ZIP genes in apple were differentially expressed among tissues and developmental stages under Zn and Fe deficiency. The overexpression of MdZIP10 increased the content of Zn and Fe in Arabidopsis thaliana L. and MdZIP10 played crucial roles in the uptake and transport of Zn and Fe. MdZIP10 was able to rescue growth of Zn2+ and Fe2+ uptake defective yeast mutants under Zn2+ and Fe2+ deficient conditions, respectively. Symptoms of Zn and Fe deficiency were alleviated in the MdZIP10 transgenic plants. The expression of genes related to Fe and Zn uptake and transport was induced in the MdZIP10 transgenic plants, thereby stimulating endogenous Fe and Zn uptake and transport mechanisms. The present study lays the foundation for future functional analysis of ZIP genes in apple.

ZmMYB31, a R2R3-MYB transcription factor in maize, positively regulates the expression of CBF genes and enhances resistance to chilling and oxidative stress.

Maize (Zea mays L.) is an important model plant with an important role in agriculture and national economies all over the world. The optimum growth temperature of maize is between 25 and 28 °C. At temperatures below 12 °C, maize is vulnerable to damage by chilling stress. MYB transcription factors play important roles in plants' response to low temperature stress. Maize ZmMYB31 encodes a R2R3-MYB transcription factor, ZmMYB31, which localized in the nucleus. ZmMYB31 expression was induced by chilling stress and the highest expression level was detected with the 24 h chilling treatment. ZmMYB31 expression also increased in overexpressing Arabidopsis lines. The minimal fluorescence (Fo) with all photosystem II reaction centers open increased in wild type (WT) and transgenic plants under chilling stress, with the highest increase in WT. The maximal photochemical efficiency of photosystem II (Fv/Fm) decreased more in WT than in transgenic plants during chilling stress. Furthermore, the ZmMYB31-overexpressing lines showed higher superoxide dismutase and ascorbate peroxidase activity and lower reactive oxygen species (ROS) content than the WT. The expression of genes related to chilling stress was higher in transgenic plants than in WT. These results suggest that ZmMYB31 plays a positive regulatory role in chilling and peroxide stress by regulating the expression of chilling stress-related genes to reduce ion extravasation, ROS content, and low-temperature photoinhibition, thereby improving low temperature resistance.

Monoterpenoid indole alkaloids from Gardneria multiflora.

Six new monoterpenoid indole alkaloids, 19(E)-9-demethoxy-16-dehydroxylchitosenine-17-O- ß-d-glucopyranoside (1), 19(E)-9,10-didemethoxy-16-dehydroxylchitosenine-17-O-ß-d-gluco-pyranoside (2), 19(E)-9,10-didemethoxy-16-dehydroxyl-11-methoxychitosenine (3), 19(E)-9,10-didemethoxy-16-dehydroxyl-11-methoxychitosenine-17-O-ß-d-glucopyranoside (4), 19(Z)-18-carboxylgardneramine (5), and 19(E)-18-demethoxygardneramine-N (4)-oxide (6), along with four known alkaloids, were isolated from Gardneria multiflora, and their structures were elucidated by spectroscopic analysis. Compounds 1, 2 and 4 are the first example of Gardneria alkaloids whose glucose units were attached to C-17. None of the compounds were cytotoxic to any of five human cancer cell lines.

The antioxidative defense system is involved in the premature senescence in transgenic tobacco (Nicotiana tabacum NC89).

BACKGROUND: α-Farnesene is a volatile sesquiterpene synthesized by the plant mevalonate (MVA) pathway through the action of α-farnesene synthase. The α-farnesene synthase 1 (MdAFS1) gene was isolated from apple peel (var. white winter pearmain), and transformed into tobacco (Nicotiana tabacum NC89). The transgenic plants had faster stem elongation during vegetative growth and earlier flowering than wild type (WT). Our studies focused on the transgenic tobacco phenotype. RESULTS: The levels of chlorophyll and soluble protein decreased and a lower seed biomass and reduced net photosynthetic rate (Pn) in transgenic plants. Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide radicals (O 2 (·-) ) had higher levels in transgenics compared to controls. Transgenic plants also had enhanced sensitivity to oxidative stress. The transcriptome of 8-week-old plants was studied to detect molecular changes. Differentially expressed unigene analysis showed that ubiquitin-mediated proteolysis, cell growth, and death unigenes were upregulated. Unigenes related to photosynthesis, antioxidant activity, and nitrogen metabolism were downregulated. Combined with the expression analysis of senescence marker genes, these results indicate that senescence started in the leaves of the transgenic plants at the vegetative growth stage. CONCLUSIONS: The antioxidative defense system was compromised and the accumulation of reactive oxygen species (ROS) played an important role in the premature aging of transgenic plants.

The antioxidative defense system is involved in the premature senescence in transgenic tobacco (Nicotiana tabacum NC89)

BACKGROUND: α-Farnesene is a volatile sesquiterpene synthesized by the plant mevalonate (MVA) pathway through the action of α-farnesene synthase. The α-farnesene synthase 1 (MdAFS1) gene was isolated from apple peel (var. white winterpearmain), and transformed into tobacco (Nicotiana tabacum NC89). The transgenic plants had faster stem elongation during vegetative growth and earlier flowering than wild type (WT). Our studies focused on the transgenic tobacco phenotype. RESULTS: The levels of chlorophyll and soluble protein decreased and a lower seed biomass and reduced net photosynthetic rate (Pn) in transgenic plants. Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide radicals (O2._) had higher levels in transgenics compared to controls. Transgenic plants also had enhanced sensitivity to oxidative stress. The transcriptome of 8-week-old plants was studied to detect molecular changes. Differentially expressed unigene analysis showed that ubiquitin-mediated proteolysis, cell growth, and death unigenes were upregulated. Unigenes related to photosynthesis, antioxidant activity, and nitrogen metabolism were downregulated. Combined with the expression analysis of senescence marker genes, these results indicate that senescence started in the leaves of the transgenic plants at the vegetative growth stage. CONCLUSIONS: The antioxidative defense system was compromised and the accumulation of reactive oxygen species (ROS) played an important role in the premature aging of transgenic plants.

[Mass Transfer Kinetics Model of Ultrasonic Extraction of Pomegranate Peel Polyphenols].

OBJECTIVE: The dynamic mathematical model of ultrasonic extraction of polyphenols from pomegranate peel was constructed with the Fick's second law as the theoretical basis. METHODS: The spherical model was selected, with mass concentrations of pomegranate peel polyphenols as the index, 50% ethanol as the extraction solvent and ultrasonic extraction as the extraction method. In different test conditions including the liquid ratio, extraction temperature and extraction time, a series of kinetic parameters were solved, such as the extraction process (k), relative raffinate rate, surface diffusion coefficient(D(S)), half life (t½) and the apparent activation energy (E(a)). RESULTS: With the extraction temperature increasing, k and D(S) were gradually increased with t½ decreasing,which indicated that the elevated temperature was favorable to the extraction of pomegranate peel polyphenols. The exponential equation of relative raffinate rate showed that the established numerical dynamics model fitted the extraction of pomegranate peel polyphenols, and the relationship between the reaction conditions and pomegranate peel polyphenols concentration was well reflected by the model. CONCLUSION: Based on the experimental results, a feasible and reliable kinetic model for ultrasonic extraction of polyphenols from pomegranate peel is established, which can be used for the optimization control of engineering magnifying production.

A new bufadienolide with cytotoxic activity from the Chinese traditional drug Ch'an Su.

AIM: To study the bufadienolides in the Chinese traditional drug "Ch'an Su" and their cytotoxic activity. METHOD: Various chromatographic techniques were used to isolate the constituents, and their structures were elucidated through physical and spectroscopic data. RESULTS: Twenty compounds were isolated, and eighteen were evaluated in vitro for their cytotoxic activity against A-549 and K-562 cells. CONCLUSION: Compound 1 (bufalin 3ß-acrylic ester) was a new bufadienolide and exhibited the most potent activity against the two tumor cell lines with IC50 values of 7.16 and 6.83 nmol · L(-1). The relationships between structure and activity are discussed.

Hernsubanine E, a new hasubanan alkaloid from Stephania hernandifolia.

A new hasubanan alkaloid, hernsubanine E (1), as well as two known compounds p-hydroxybenzaldehyde (2) and (-)-syringaresinol (3) have been isolated from the whole plants of Stephania hernandifolia by various column chromatographic methods. Their structures were identified by physicochemical properties and spectral analyses. Compounds 2 and 3 were isolated from the genus of Stephania for the first time.