Range of factors in the reduction of hyperhydricity associated with in vitro shoots of Salvia santolinifolia Bioss / Gama de fatores na redução da hiperidricidade associada a brotos in vitro de Salvia santolinifolia Bioss

Hyperhydricity is a serious physiological disorder and affects In vitro propagation of many plants and as well of Salvia santolinifolia. The donor material to initiate the in vitro culture was the callus taken from the in vitro shoots produced on Murashig and Skoogs (MS) medium at 4.0 mg/l BA. This callus formed numerous hyperhydric shoots on culturing upon the medium of the same composition. The aim was to systematically evaluate the effect of cytokinins (Benzyladnine (BA) and N6-(-2-isopentenyl) adenine (2iP), culture vessels magnitude, medium solidification, source of nitrogen and calcium chloride for the alleviation of hyperhydricity. In the tissue cultures of S. santolinifolia BA and 2iP induced severe hyperhydricity, when other factors i.e. culture vessels magnitude and a suitable concentration of agar, ammonium nitrate (NH4NO3), potassium nitrate (KNO3) & calcium chloride (CaCl2.2H2O) were not optimized. After 30 days' culture, we observed 83.82% hyperhydric shoots at increased level (1.5 mg/l 2iP) and 81.59% at decreased levels (1.0 mg/l 2iP). On the other hand, hyperhydricity percentage at decreased (0.4%) and at increased (0.8%) levels of agar were 72.37% and 39.08%, respectively. MS medium modification with NH4NO3 (412 mg/l), KNO3 (475 mg/l) and CaCl2.2H2O (880 mg/l) was found the best medium to reduced hyperhydricity (23.6%).

A hiperidricidade é um distúrbio fisiológico sério e afeta a propagação in vitro de muitas plantas e também da Salvia santolinifolia. O material doador para iniciar a cultura in vitro foi o calo retirado dos brotos in vitro produzidos em meio Murashig e Skoogs (MS) a 4,0 mg / l BA. Esse calo formou numerosos rebentos hiperídricos em cultura no meio da mesma composição. O objetivo foi avaliar sistematicamente o efeito das citocininas (Benziladnina (BA) e N6 - (- 2-isopentenil) adenina (2iP), magnitude dos vasos de cultura, solidificação do meio, fonte de nitrogênio e cloreto de cálcio para o alívio da hiperidricidade. culturas de tecidos de S. santolinifolia BA e 2iP induziram hiperidricidade severa, quando outros fatores, como magnitude dos vasos de cultura e uma concentração adequada de ágar, nitrato de amônio (NH4NO3), nitrato de potássio (KNO3) e cloreto de cálcio (CaCl2.2H2O), não foram otimizados. Após 30 dias de cultura, observamos 83,82% de brotos hiperídricos em níveis aumentados (1,5 mg / l 2iP) e 81,59% em níveis reduzidos (1,0 mg / l 2iP). Por outro lado, a porcentagem de hiperidricidade diminuiu (0,4%) e em níveis aumentados (0,8%) de ágar foram 72,37% e 39,08%, respectivamente. A modificação do meio MS com NH4NO3 (412 mg / l), KNO3 (475 mg / l) e CaCl2.2H2O (880 mg / l) foi encontrada melhor hiperidricidade média a reduzida (23,6%).

[Simultaneous determination of 40 plant growth regulators, fungicides, insecticides, and antibiotics in bean sprouts by QuEChERS-high performance liquid chromatography-tandem mass spectrometry].

Chromatography combined with mass spectrometry is the most commonly used detection technology, and it offers the advantages of high sensitivity and high selectivity. The quick, easy, inexpensive, effective, rugged, and safe (QuEChERS) method is low-cost, effective, and time efficient. The application of the QuEChERS has now been extended to the analysis of contaminants in food samples. The aim of the study was to identify different concentration levels of multiple harmful drug residues in bean sprouts. In this study, QuEChERS coupled with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was established for the simultaneous determination of 40 plant growth regulators, fungicides, insecticides, and antibiotics in bean sprouts. In the HPLC-MS/MS experiment, gibberellic acid, 4-fluorophenoxyacetic acid, chloramphenicol, N6-(δ2-isopentenyl)-adenine, 6-benzylaminopurine, 4-chlorophenoxyacetic acid, and 2,4-dichlorophenoxyacetic acid (2,4-D) were analyzed by MS/MS with negative electrospray ionization (ESI-). The other 33 target analytes (chlormequat, ronidazole, metronidazole, pymetrozine, dimetridazole, methomyl, carbendazim, enoxacin, levofloxacin, pefloxacin mesylate, norfloxacin, ciprofloxacin, enrofloxacin, thiabendazole, lomefloxacin, chlorpyrifos, sarafloxacin, imidacloprid, etc.) were analyzed by MS/MS with positive electrospray ionization (ESI+). Sensitive MS conditions were realized by optimizing the instrumental parameters such as the desolvent temperature, collision energy, spraying needle position, precursor ions, and product ions. Then, the optimal pretreatment method was determined by comparing the recovery rates of the 40 drugs obtained with different extraction solvents (methanol, acetonitrile, acetonitrile containing 0.1% ammonia, acetonitrile with 1% acetic acid), different extraction methods (ultrasonic extraction, shaking extraction), and purification with primary secondary amine (PSA) and C18. In this study, the bean sprouts samples were extracted twice by 10 mL acetonitrile with 1% acetic acid, and extracted under ultrasonic conditions. Then, the extracting solution was only cleaned with 100 mg C18. The chromatographic separation of the 40 compounds was accomplished on a Waters ACQUITY UPLC BEH C18 column (100 mm×2.1 mm, 1.7 µm) with gradient elution. Methanol and 0.01% formic acid aqueous solution were used as the mobile phases. The 40 compounds were analyzed in the multiple reaction monitoring (MRM) mode. The matrix matching external standard method was used for quantitative determination. The results showed that the 40 compounds could be analyzed within 15 min. Under the optimized conditions, the calibration curves showed good linearities for the 40 compounds, and the coefficients of determination (r2) were greater than 0.99 in the range of 2-200 µg/L. The limits of detection (LODs) and limits of quantification (LOQs) were in the range of 0.1-3 µg/kg and 0.3-9 µg/kg, respectively. Using negative bean sprouts as the substrates, the recovery tests were carried out at three spiked levels of 5, 10, and 50 µg/kg. The average recoveries of the 40 drugs were 78.5% to 115.3%, and the corresponding relative standard deviations (RSDs) were 1.3% to 9.7% (n=6). This method was successfully applied to the analysis of the 40 drug residues in 21 batches of local bean sprouts in Handan city. The results revealed the presence of extensive drug residues in the bean sprouts. The 26 batches were detected to varying degrees, among which 4-chlorophenoxyacetic acid, carbendazim, 6-benzyladenine, 2,4-D, enrofloxacin, and metronidazole were detected at high rates. The detection rates of 4-chlorophenoxyacetic acid, 6-benzyladenine, carbendazim, 2,4-D, gibberellic acid, and enrofloxacin were 28.6%, 19.0%, 9.5%, 9.5%, 4.8%, and 4.8%, respectively. The contents ranged from 37.5-352.4, 32.4-273.1, 28.8-38.7, 316.1-20.2, 19.9 and 13.6 µg/kg, respectively. Given its advantages of simplicity, rapidness, and high sensitivity, the developed method can be used for the rapid and accurate determination of trace levels of the 40 drug residues in large quantities of bean sprouts.

Growth regulators and their reflection on different hop genotypes cultivated under in vitro conditions / Reguladores de crescimento e seu reflexo em diferentes genótipos de lúpulo cultivados in vitro

Hops is a new culture in Brazil. Tissue culture can be an important technique for rapid hop propagation. This paper aims to characterize responses from different genotypes under different growth regulators through the interrelationship of response variables important to hop in vitro growth. Three genotypes were cultivated in six culture media with different combinations of growth regulators, BAP (6-benzylaminopurine), IAA (3-indolacetic acid) and GA3 (gibberellic acid). The means were compared by orthogonal contrasts and the interrelationship of the response variables was performed by path analysis. American genotypes showed favorable root development under the BAP + IAA combination, while the use of IAA improved shoot development. The origin of genotypes was important for defining the best protocol for in vitro cultivation. The path coefficient showed that the variable number of shoots has stronger direct effect on the number of nodal segments. Additionally, in tissue culture assays, the use of a covariable and proper error distribution significantly increased experimental accuracy.

O lúpulo é uma nova cultura no Brasil. A cultura de tecidos pode ser uma técnica importante para a propagação rápida do lúpulo. Este artigo tem como objetivo caracterizar respostas de diferentes genótipos sob diferentes reguladores de crescimento através da inter-relação de variáveis de resposta importantes para o crescimento in vitro. Três genótipos foram cultivados em seis meios de cultura com diferentes combinações de reguladores de crescimento, BAP (6-benzilaminopurina), AIA (ácido 3-indolacético) e GA3 (ácido giberélico). As médias foram comparadas por contrastes ortogonais e a inter-relação das variáveis de resposta foi realizada por análise de trilha. Os genótipos americanos apresentaram desenvolvimento radicular favorável sob a combinação BAP + AIA, enquanto o uso do AIA melhorou o desenvolvimento da parte aérea. A origem dos genótipos foi importante para definir o melhor protocolo para o cultivo in vitro. O coeficiente de trilha mostrou que a variável número de brotos tem um efeito direto mais forte no número de segmentos nodais. Adicionalmente, em experimentos com cultura de tecidos, o uso de uma covariável e distribuição de erro adequada aumentou significativamente a precisão experimental.

The application of ammonium sulphate and amino acid on cotton: effects on can improve growth, yield, quality and nitrogen absorption / Aplicação de sulfato de amônio e aminoácido no algodão: efeitos sobre o crescimento, o rendimento, a qualidade e a absorção de nitrogênio

A field study was carried out to determine the influence of foliage applied plant growth promoter and retardant in improving soil applied sulphur fertilizer use efficiency in cotton during two consecutive summers 2014 and 2015. Experimental trial comprised of three different sources of sulphur (ammonium sulphate, potassium sulphate and elemental sulphur) and foliar spray of plant growth promoter and growth retardant including tap water was taken as control. Among treatments soil applied ammonium sulphate with foliage applied amino acid produced maximum plant height, sympodial branches, pods per plant, seed cotton yield, fiber yield, biological yield, protein contents, oil contents and leaf nitrogen uptake as compared to the other treatments. Whereas, soil applied potassium sulphate with foliar spray of mepiquat chloride on cotton significantly improved the boll weight and leaf potassium uptake. We conclude that soil applied ammonium sulphate and foliage spray of amino acid was more effective in improving the productivity and quality attributes of cotton.

Foi realizado um estudo de campo para determinar a influência do promotor de crescimento das plantas e retardador da folhagem em algodão, para melhora da eficiência do uso de fertilizantes à base de enxofre aplicados no solo durante dois verões consecutivos (2014 e 2015). O ensaio experimental foi composto de três fontes diferentes de enxofre (sulfato de amônio, sulfato de potássio e enxofre elementar) e pulverização foliar do promotor de crescimento de plantas e retardador de crescimento, incluindo água da torneira que foi tomada como controle. Entre os tratamentos, o sulfato de amônio aplicado no solo com aminoácido aplicado na folhagem produziu o máximo na altura da planta, ramos simodiais, capulhos por planta, rendimento de algodão em caroço, rendimento de fibra, rendimento biológico, conteúdo de proteínas, conteúdo de óleo e absorção de nitrogênio nas folhas quando comparado a outros tratamentos. Enquanto o solo fertilizado com sulfato de potássio e aplicação foliar de cloreto de mepiquat no algodão melhorou, significativamente, o peso do capulho e a absorção de potássio nas folhas. Sulfato de amônio aplicado no solo e a aplicação foliar de aminoácidos foram mais eficazes na melhora dos atributos de produtividade e qualidade do algodão.

Sumatran fleabane (Conyza sumatrensis [Retz.] E. Walker) control in soybean with combinations of burndown and preemergence herbicides applied in the off-season

Sumatran fleabane (Conyza sumatrensis [Retz.] E. Walker) can be found in many different agricultural environments and impact different crops, such as soybeans and corn. It is believed that the application of burndown and preemergence herbicides in the off-season are effective in controlling Sumatran fleabane in soybean crops. The objective was to evaluate the effectiveness of burndown and preemergence herbicides in the off-season, with one or two applications, in the control of Sumatran fleabane in soybean cultivation. Five field experiments were conducted in Maripá, state of Paraná (PR), Brazil. The treatments consisted of the application of burndown herbicides in combinations with preemergence ones, with one or two applications. Control of Sumatran fleabane and soybean yield were evaluated. With the set of experiments, it is highlighted that the strategy combining more applications, with different herbicides, burndown and preemergence, is more promising in the control of Sumatran fleabane. When comparing synthetic auxins, dicamba and triclopyr stand out. For sequential application, worse performance was observed for diquat. Combinations between burndown and preemergence herbicides were effective in controlling Sumatran fleabane, for pre sowing application in soybean. With emphasis on managements with sequential applications of saflufenacil with glufosinate or glyphosate. The strategy combining more applications, with different herbicides, burndown and preemergence herbicides, is more promising in the control of Sumatran fleabane.

In vivo FRET-FLIM reveals ER-specific increases in the ABA level upon environmental stresses.

Plant hormone abscisic acid (ABA) is essential for regulating plant growth and various stress responses. ABA-mediated signaling depends on local ABA levels rather than the overall cellular ABA concentration. While cellular concentration of ABA can be detected using Förster resonance energy transfer (FRET)-based ABA probes, direct imaging of subcellular ABA levels remains unsolved. Here, we modified the previously reported ABAleon2.1 and generated a new ABA sensor, named ABAleon2.1_Tao3. Via transient expression in tobacco (Nicotiana tabacum) protoplasts, we targeted ABAleon2.1_Tao3s to the endoplasmic reticulum (ER) membrane with the ABA sensing unit facing the cytosol and the ER, respectively, through a nanobody-epitope-mediated protein interaction. Combining FRET with fluorescence lifetime imaging microscopy, ABA-triggered-specific increases in the fluorescence lifetime of the donor mTurquoise in the ABAleon2.1_Tao3 were detected in both transient assays and stably transformed Arabidopsis plants. In tobacco protoplasts, ER membrane-targeted ABAleon2.1_Tao3s showed a generally higher basal level of ABA in the ER than that in the cytosol and ER-specific alterations in the level of ABA upon environmental cues. In ABAleon2.1_Tao3-transformed Arabidopsis roots, mannitol triggered increases in cytosolic ABA in the division zone and increases in ER ABA in the elongation and maturation zone within 1 h after treatment, both of which were abolished in the bg1-2 mutant, suggesting the requirement for BG1 in osmotic stress-triggered early ABA induction in Arabidopsis roots. These data demonstrate that ABAleon2.1_Tao3s can be used to monitor ABA levels in the cytosol and the ER, providing key information on stress-induced changes in the level of ABA in different subcellular compartments.

Simultaneous determination of 16 important biologically active phytohormones in Dendrobium huoshanese by pressurized capillary electrochromatography.

A rapid pressurized capillary electrochromatography (pCEC) method has been successfully developed for the simultaneous determination of 16 phytohormones in Dendrobium huoshanense. Effects of wavelength, mobile phase, the flow rate, pH value, concentration of buffer and applied voltage were investigated, respectively. The results showed that the 16 phytohormones could be baseline-separated rapidly in less than 21 min on a reversed-phase EP-100-20/45-3-C18 capillary column (total length of 45 cm, effective length of 20 cm, diameter of 100 µm, ODS packing inside for 3 µm) with ACN/5.0 mM ammonium acetate (containing 0.05% formic acid, pH = 3) as the mobile phase using gradient elution mode as follows: 0.1-10.0 min 40%ACN,10-15.0 min 70%ACN, 15.0-20 min 80% ACN, 20-21.0 min 80% ACN at a flow rate of 0.12 mL/min, applied voltage of -5 kV and a UV detection wavelength of 210 nm. The method validation howed that the established method is precise and stability, and the RSDs of intra- and inter-day precision based retention time and peak area were all below 5%. Employed the established method, in our experimental conditions, total 6 endogenous hormones including IAA, IBA, NAA, GA, ABA, t-Z were detected in D. huoshense. However, a relative larger amount of exogenous hormone 2,4-D (25.3 ~ 4.2 µg/kg) and 6-BA (79.5 ~ 35.4 µg/kg) were detected in 1 ~ 4 year old cultivated D. huoshense, suggesting there were still a certain amount of exogenous hormone residue in tissue-cultured D. huoshanese though they had been transplanted to field cultivation from the test-tube plantlets for several years.

Development, validation, and application of a multi-method for the determination of mycotoxins, plant growth regulators, tropane alkaloids, and pesticides in cereals by two-dimensional liquid chromatography tandem mass spectrometry.

Mycotoxins and pesticides regularly co-occur in agricultural products worldwide. Thus, humans can be exposed to both toxic contaminants and pesticides simultaneously, and multi-methods assessing the occurrence of various food contaminants and residues in a single method are necessary. A two-dimensional high performance liquid chromatography tandem mass spectrometry method for the analysis of 40 (modified) mycotoxins, two plant growth regulators, two tropane alkaloids, and 334 pesticides in cereals was developed. After an acetonitrile/water/formic acid (79:20:1, v/v/v) multi-analyte extraction procedure, extracts were injected into the two-dimensional setup, and an online clean-up was performed. The method was validated according to Commission Decision (EC) no. 657/2002 and document N° SANTE/12682/2019. Good linearity (R2 > 0.96), recovery data between 70-120%, repeatability and reproducibility values < 20%, and expanded measurement uncertainties < 50% were obtained for a wide range of analytes, including very polar substances like deoxynivalenol-3-glucoside and methamidophos. However, results for fumonisins, zearalenone-14,16-disulfate, acid-labile pesticides, and carbamates were unsatisfying. Limits of quantification meeting maximum (residue) limits were achieved for most analytes. Matrix effects varied highly (-85 to +1574%) and were mainly observed for analytes eluting in the first dimension and early-eluting analytes in the second dimension. The application of the method demonstrated the co-occurrence of different types of cereals with 28 toxins and pesticides. Overall, 86% of the samples showed positive findings with at least one mycotoxin, plant growth regulator, or pesticide.

Selective, sensitive, and miniaturized analytical method based on molecularly imprinted graphene oxide composites for the determination of naphthalene-derived plant growth regulators in apples.

This paper reports a selective, sensitive, and miniaturized analytical method based on a molecularly imprinted graphene oxide (MIP-GO) composite as adsorbent for miniaturized tip solid-phase extraction (MTSPE) to determine naphthalene-derived plant growth regulators (PGRs) in apples. The proposed method combines the advantages of MIP-GOs (high selectivity), MTSPE (low consumption), and high-performance liquid chromatography-fluorescence detection (high sensitivity). Under optimized conditions, the method exhibited appreciable linearity (2.00-200 ng/g), low detection limits (0.21-0.53 ng/g), high accuracy (absolute recoveries: 87.6-99.5%), and high precision (relative standard deviations ≤ 3.0%), along with low consumption (0.5 mL sample solution and 2.0 mg adsorbent). In addition, the adsorption performance of the MIP-GO adsorbent did not decrease over ten months, highlighting the long storage and operational lifetime of the adsorbent. The proposed method was employed for the analysis of naphthalene-derived PGR residues in apples and exhibited promising potential for application in food safety analysis.

A Tailored High-Efficiency Sample Pretreatment Method for Simultaneous Quantification of 10 Classes of Known Endogenous Phytohormones.

One of the hottest topics in plant hormone biology is the crosstalk mechanisms, whereby multiple classes of phytohormones interplay with each other through signaling networks. To better understand the roles of hormonal crosstalks in their complex regulatory networks, it is of high significance to investigate the spatial and temporal distributions of multiple -phytohormones simultaneously from one plant tissue sample. In this study, we develop a high-sensitivity and high-throughput method for the simultaneous quantitative analysis of 44 phytohormone compounds, covering currently known 10 major classes of phytohormones (strigolactones, brassinosteroids, gibberellins, auxin, abscisic acid, jasmonic acid, salicylic acid, cytokinins, ethylene, and polypeptide hormones [e.g., phytosulfokine]) from only 100 mg of plant sample. These compounds were grouped and purified separately with a tailored solid-phase extraction procedure based on their physicochemical properties and then analyzed by LC-MS/MS. The recoveries of our method ranged from 49.6% to 99.9% and the matrix effects from 61.8% to 102.5%, indicating that the overall sample pretreatment design resulted in good purification. The limits of quantitation (LOQs) of our method ranged from 0.06 to 1.29 pg/100 mg fresh weight and its precision was less than 13.4%, indicating high sensitivity and good reproducibility of the method. Tests of our method in different plant matrices demonstrated its wide applicability. Collectively, these advantages will make our method helpful in clarifying the crosstalk networks of phytohormones.

Changes in the distribution of endogenous hormones in Phyllostachys edulis 'Pachyloen' during bamboo shooting.

In this study, we investigated the changes in the distribution and regulation of endogenous hormones in Phyllostachys edulis 'Pachyloen' during bamboo shooting. Enzyme-linked immunosorbent assay was used to measure the mass fractions of indole-3-acetic acid (IAA), gibberellic acid (GA), zeatin riboside (ZR), and abscisic acid (ABA) in rhizomes, shoots, and maternal bamboo organs during shoot sprouting, shoot growth, and new-bamboo formation. Measurements were compared among bamboo parts and developmental periods. The overall mass fractions of IAA and ABA were significantly higher than those of ZR and GA, driven by differences among bamboo parts and developmental periods. The abundance of each endogenous hormone varied among bamboo parts and developmental periods. During bamboo shooting, ABA had the highest mass fraction in all bamboo parts sampled, followed by IAA, GA, and ZR. Among bamboo parts, rhizomes had more IAA, ZR, and GA than the other parts, but significantly less ABA. Winter shoots had higher ZR: IAA and GA: IAA ratios than rhizomes and maternal bamboo organs. During shoot growth, ABA was the most abundant hormone in rhizomes and maternal bamboo organs, followed by IAA, ZR, and GA. In contrast, IAA was the most abundant hormone in spring shoots, followed by ABA, ZR, and GA. Maternal bamboo organs had a significantly higher ZR: GA ratio, and significantly lower IAA: ABA, ZR: ABA, and GA: ABA ratios than rhizomes. Spring shoots had significantly higher IAA: ABA, ZR: ABA, and GA: ABA ratios than rhizomes and maternal bamboo organs; significantly higher ZR mass fractions, and ZR: GA and ZR: IAA ratios and significantly lower ABA mass fractions than rhizomes; and significantly higher GA: IAA ratio than maternal bamboo organs. During new-bamboo formation, ABA was the most abundant hormone in rhizomes, winter shoots, and maternal bamboo organs, followed by IAA, ZR, and GA. Maternal bamboo organs had significantly lower IAA mass fractions and significantly higher ABA mass fractions than rhizomes and new bamboo tissue. IAA and ABA abundances exhibited an inverse relationship in rhizomes and maternal bamboo organs. GA: ABA and GA: IAA ratios decreased gradually and other hormone ratios exhibited parabolic trends over the bamboo-shooting period, with the highest ratios observed in new bamboo tissues. Overall, the coordination or antagonism among endogenous hormones plays a key regulatory role in bamboo shoot growth. The formation of thick walls in P. edulis 'Pachyloen', one of its major traits, may be partially attributed to the relatively high IAA and ZR and low GA mass fractions.

Phytohormone production by the arbuscular mycorrhizal fungus Rhizophagus irregularis.

Arbuscular mycorrhizal symbiosis is a mutualistic interaction between most land plants and fungi of the glomeromycotina subphylum. The initiation, development and regulation of this symbiosis involve numerous signalling events between and within the symbiotic partners. Among other signals, phytohormones are known to play important roles at various stages of the interaction. During presymbiotic steps, plant roots exude strigolactones which stimulate fungal spore germination and hyphal branching, and promote the initiation of symbiosis. At later stages, different plant hormone classes can act as positive or negative regulators of the interaction. Although the fungus is known to reciprocally emit regulatory signals, its potential contribution to the phytohormonal pool has received little attention, and has so far only been addressed by indirect assays. In this study, using mass spectrometry, we analyzed phytohormones released into the medium by germinated spores of the arbuscular mycorrhizal fungus Rhizophagus irregularis. We detected the presence of a cytokinin (isopentenyl adenosine) and an auxin (indole-acetic acid). In addition, we identified a gibberellin (gibberellin A4) in spore extracts. We also used gas chromatography to show that R. irregularis produces ethylene from methionine and the α-keto γ-methylthio butyric acid pathway. These results highlight the possibility for AM fungi to use phytohormones to interact with their host plants, or to regulate their own development.

Indoleacetic Acid Levels in Wheat and Rice Seedlings under Oxygen Deficiency and Subsequent Reoxygenation.

The lack of oxygen and post-anoxic reactions cause significant alterations of plant growth and metabolism. Plant hormones are active participants in these alterations. This study focuses on auxin-a phytohormone with a wide spectrum of effects on plant growth and stress tolerance. The indoleacetic acid (IAA) content in plants was measured by ELISA. The obtained data revealed anoxia-induced accumulation of IAA in wheat and rice seedlings related to their tolerance of oxygen deprivation. The highest IAA accumulation was detected in rice roots. Subsequent reoxygenation was accompanied with a fast auxin reduction to the control level. A major difference was reported for shoots: wheat seedlings contained less than one-third of normoxic level of auxin during post-anoxia, while IAA level in rice seedlings rapidly recovered to normoxic level. It is likely that the mechanisms of auxin dynamics resulted from oxygen-induced shift in auxin degradation and transport. Exogenous IAA treatment enhanced plant survival under anoxia by decreased electrolyte leakage, production of hydrogen peroxide and lipid peroxidation. The positive effect of external IAA application coincided with improvement of tolerance to oxygen deprivation in the 35S:iaaM × 35S:iaaH lines of transgene tobacco due to its IAA overproduction.

Hormone profile changes occur in roots and leaves of Micro-Tom tomato plants when exposing the aerial part to low doses of UV-B radiation.

During the last decades, many studies investigated the effects of UV-B on the above-ground organs of plants, directly reached by the radiation but, to the best of our knowledges, the influence of mild UV-B doses on root hormones was not explored. Consequently, this research aimed at understanding whether low, not-stressful doses of UV-B radiation applied above-ground influenced the hormone concentrations in leaves and roots of Micro-Tom tomato (Solanum lycopersicum L.) plants during 11 days of treatment and after 3 days of recovery. In particular, ethylene, abscisic acid, jasmonic acid, salicylic acid and indoleacetic acid were investigated. The unchanged levels of chlorophyll a and b, lutein, total xanthophylls and carotenoids, as well as the similar H2O2 concentration between control and treated groups suggest that the UV-B dose applied was well tolerated by the plants. Leaf ethylene emission decreased after 8 and 11 days of irradiation, while no effect was found in roots. Conversely, indoleacetic acid underwent a significant reduction in both organs, though in the roots the decrease occurred only at the end of the recovery period. Salicylic acid increased transiently in both leaves and roots on day 8. Changes in leaf and root hormone levels induced by UV-B radiation were not accompanied by marked alterations of plant architecture. The results show that irradiation of above-ground organs with low UV-B doses can affect the hormone concentrations also in roots, with likely implications in stress and acclimation responses mediated by these signal molecules.

An artificial metalloenzyme biosensor can detect ethylene gas in fruits and Arabidopsis leaves.

Enzyme biosensors are useful tools that can monitor rapid changes in metabolite levels in real-time. However, current approaches are largely constrained to metabolites within a limited chemical space. With the rising development of artificial metalloenzymes (ArM), a unique opportunity exists to design biosensors from the ground-up for metabolites that are difficult to detect using current technologies. Here we present the design and development of the ArM ethylene probe (AEP), where an albumin scaffold is used to solubilize and protect a quenched ruthenium catalyst. In the presence of the phytohormone ethylene, cross metathesis can occur to produce fluorescence. The probe can be used to detect both exogenous- and endogenous-induced changes to ethylene biosynthesis in fruits and leaves. Overall, this work represents an example of an ArM biosensor, designed specifically for the spatial and temporal detection of a biological metabolite previously not accessible using enzyme biosensors.

Effects of exogenous Ca2+ on phenolic accumulation and physiological changes in germinated wheat (Triticum aestivum L.) under UV-B radiation.

Exogenous Ca2+ affects the phenolic metabolism and physiological indices of germinated wheat under ultraviolet-B (UV-B) radiation, but the mechanism is unclear. The current study applied exogenous Ca2+ and Ca2+ channel blocker LaCl3 to the germinated wheat under UV-B radiation to unravel the role of Ca2+. The results indicated that total phenolic content (TPC) of the 4-day germinated wheat under UV-B radiation with CaCl2 (UV-B+Ca) treatment significantly increased by 10.3% as compared to the UV-B treatment. Gene expression levels of p-coumarate 3-hydroxylase, cinnamic acid 4-hydroxylase and caffeic acid O-methyltransferase were positively correlated with the content of ferulic and p-coumaric acids, respectively. Exogenous Ca2+ could significantly alleviate the membrane lipid peroxidation, activate the antioxidant enzymes and regulate the phytohormone level under UV-B radiation. This study suggested that exogenous Ca2+ participated in the phenolic metabolism and physiological regulation in germinated wheat under UV-B radiation.

Multi-residue analysis of plant growth regulators and pesticides in traditional Chinese medicines by high-performance liquid chromatography coupled with tandem mass spectrometry.

A multi-residue method was developed for the simultaneous determination of 24 plant growth regulators (PGRs) and 11 representative pesticides that were widely applied in plants used in traditional Chinese medicines (TCMs) by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The method was validated taking into consideration EU guidelines; calibration curves for all of the targeted analytes showed correlation coefficients (γ2) higher than 0.9901. The limits of detection (LOD) ranged from 0.2 to 8 µg/kg. The average recovery for all analytes in spiked samples ranged from 63.18 to 127.23%, with a relative standard deviation of ≤ 15%. The proposed method has been applied to 480 batches of TCM samples, including 34 species of medicinal plants, from the TCM market. The results showed that 14 PGRs and 5 pesticides were detected, including choline chloride, chlormequat, paclobutrazol, uniconazole, phoxim, etc. Among them, there were high detection rates for chlormequat (40%), choline chloride (100%), atonik (73.75%), abscisic acid (80.83%), and indole-3-acetic acid (41.25%). The residual level of paclobutrazol in Ophiopogonis radix exceeded the recommended maximum residue limits (MRLs) according to GB 2763-2016. In addition, 14 agrochemicals used in TCM planting were collected and detected; the result showed various PGRs were detected in samples registered as fertilizer. These results indicate that PGRs and pesticides were widely used in the cultivation of medicinal plants, especially for radix and rhizome herbs. The residue of targeted PGRs and pesticides in TCM samples from this study have a high frequency and high level. Graphical abstract.

Ethylene Measurements from Sweet Fruits Flowers Using Photoacoustic Spectroscopy.

Ethylene is a classical plant hormone and has appeared as a strong molecule managing many physiological and morphological reactions during the life of a plant. With laser-based photoacoustic spectroscopy, ethylene can be identified with high sensitivity, at a high rate and with very good selectivity. This research presents the dynamics of trace gases molecules for ethylene released by cherry flowers, apple flowers and strawberry flowers. The responses of distinctive organs to ethylene may fluctuate, depending on tissue sensitivity and the phase of plant development. From the determinations of this study, the ethylene molecules at the flowers in the nitrogen flow were established in lower concentrations when the value is correlated to the ethylene molecules at the flowers in synthetic air flow.

Simultaneous determination of pesticides, mycotoxins, tropane alkaloids, growth regulators, and pyrrolizidine alkaloids in oats and whole wheat grains after online clean-up via two-dimensional liquid chromatography tandem mass spectrometry.

In this study, a two-dimensional liquid chromatography tandem mass spectrometry method was developed and validated for the determination of pesticide residues and contaminants in whole wheat grains and oats. The samples were extracted with a mixture of acetonitrile and water and were injected into the two-dimensional LC-MS/MS system without any further clean-up or sample preparation. Samples were analyzed with four different matrix matched calibrations. Matrix effects were evaluated by comparing analyte signals in the respective matrix matched standard with the neat solvent standards. The final method was validated according to the current Eurachem validation guide and SANTE document. The number of successfully validated analytes throughout all three validation levels in oats and wheat, respectively, were as follows: 330 and 316 out of 370 pesticides, 6 and 13 out of 18 pyrrolizidine alkaloids and 7 out of 9 regulated mycotoxins. Moreover, both plant growth regulators mepiquat and chlormequat as well as the tropane alkaloids atropine and scopolamine met the validation criteria. The majority of pesticides showed limits of detection below 1 µg kg-1, pyrrolizidine alkaloids below 0.7 µg kg-1, tropane alkaloids below 0.2 µg kg-1, growth regulators below 0.7 µg kg-1 and mycotoxins below 8 µg kg-1 in both matrices.

Rapid and Visual Detection and Quantitation of Ethylene Released from Ripening Fruits: The New Use of Grubbs Catalyst.

Herein, we report on fluorophore-tagged Grubbs catalysts as turn-on fluorescent probes for the sensitive detection and quantitation of ethylene, a plant hormone that plays a critical role in many phases of plant growth and fruit ripening. The ruthenium-based weak fluorescent probes were prepared handily through the metathesis reaction between the first-generation Grubbs catalyst and selected fluorophores that have high quantum yields and contain terminal vinyl groups. Upon exposure to ethylene, fluorescence enhancement was observed via the release of fluorophore from the probe. Our probe shows an excellent limit of detection for ethylene at 0.9 ppm in air and was successfully applied for monitoring ethylene released during the fruit-ripening process. Our work opens up a new avenue of application of Grubbs catalysts for bioanalytical chemistry of ethylene, which is critically important in plant biology, agriculture, and food industry.