Suspension-cultured Tomato Cells Research Articles

Production of chitinase in elicited tomato cell suspensions and its application as a biopesticide and fungicide against soil-borne pests and fungi

Soil-borne fungi and pests present significant challenges to crop production, causing diseases and reducing yields. This study explores the potential of producing chitinase from tomato cell suspension cultures and its application in controlling soil-borne fungi and pests. Tomato cell suspension cultures were established from seeds, and different elicitors—including chitin, fungal debris, and fungal metabolites—were used to optimize chitinase production. The enzyme was purified, and its physicochemical properties were characterized. Its antifungal activity was tested against various soil-borne fungi, while termiticidal activity was evaluated using four bioassay methods. Additionally, the nematicidal activity of chitinase was assessed against different stages of root-knot nematodes. Results indicated that chitin media induced the highest chitinase production and cell growth compared to other elicitors. The purified chitinase had a molecular weight of 34 kDa, with a maximum velocity (Vmax) of 0.940 μg NAG/min and a Michaelis constant of 1.236 mg colloidal chitin/mL. The turnover number (Kcat) was 1.598 μg NAG/min/mol chitinase. The enzyme exhibited a broad range of activity and stability across varying pH levels and temperatures, as well as remarkable stability under different storage times and in the presence of metal ions. Tomato-derived chitinase demonstrated superior antifungal, nematicidal, and termiticidal activities compared to common chemical fungicides and pesticides. Chitinase was successfully produced from tomato cell suspension culture in this study, highlighting its potential as a biocontrol agent for sustainable pest and disease management in agriculture.

Elicitation and precursor induced approaches for the enhancement of α−tocopherol production using suspension cultures of Solanum lycopersicum

Elicitation and precursor feeding are the effective strategies for enhancing the synthesis of bioactive compounds in plant cell suspension cultures. The present study aimed to explore an efficient elicitation and precursor feeding protocol and its effect on inducing the accumulation of α-tocopherol in Solanum lycopersicum (tomato) suspension cell culture. The tomato cell suspension cultures were treated with different elicitors (Methyl Jasmonate, Salicylic acid and Yeast extract) and precursors (Homogentisic acid, Tyrosine, Hydroxypyruvic acid and Phytol) and the effect of α-tocopherol production was studied. Significant increase in the α-tocopherol was observed on day 5 upon methyl jasmonate treatment which represented 17.7 fold increase in comparison to the control. The treatment of precursor in combination viz., 150 μM Homogentisic acid + 150 μM Phytol showed the maximum enhancement of α-tocopherol up to 22 fold on day 10 compared to the untreated control. These results suggested that the suspension cultures combining with the optimal precursor feeding and elicitors enhanced the production of α-tocopherol in economically important tomato cell cultures.

Growth Dynamics and Cell Viability in Tomato Suspension Cultures Derived from Different Types of Calli

To establish a dynamic and fine suspension culture, four different methods of tomato cell suspension culture were compared. Hypocotyl explants of the tomato cultivar Jina were used for callus induction on Murashige and Skoog (MS) Medium supplemented with three different phytohormone combinations. Then, one gram of each type of calli was transferred to 50 mL of liquid MS medium with four combinations of auxins and cytokinins to produce cell suspensions. The growth rate, judged by cell turbidity, cell fresh weight, and cell viability was evaluated. The best suspension culture was obtained by using friable calli formed on MS medium containing 1 mg L-1 NAA and 0.1 mg L-1 kinetin, transferred to the liquid MS supplemented with 2 mg L-1 NAA, 0.2 mg L-1 2, 4-D and 0.2 mg L-1 zeatin.

The Systemin Signaling Cascade As Derived from Time Course Analyses of the Systemin-responsive Phosphoproteome*[S

Systemin is a small peptide with important functions in plant wound response signaling. Although the transcriptional responses of systemin action are well described, the signaling cascades involved in systemin perception and signal transduction at the protein level are poorly understood. Here we used a tomato cell suspension culture system to profile phosphoproteomic responses induced by systemin and its inactive Thr17Ala analog, allowing us to reconstruct a systemin-specific kinase/phosphatase signaling network. Our time-course analysis revealed early phosphorylation events at the plasma membrane, such as dephosphorylation of H+-ATPase, rapid phosphorylation of NADPH-oxidase and Ca2+-ATPase. Later responses involved transient phosphorylation of small GTPases, vesicle trafficking proteins and transcription factors. Based on a correlation analysis of systemin-induced phosphorylation profiles, we predicted substrate candidates for 44 early systemin-responsive kinases, which includes receptor kinases and downstream kinases such as MAP kinases, as well as nine phosphatases. We propose a regulatory module in which H+-ATPase LHA1 is rapidly de-phosphorylated at its C-terminal regulatory residue T955 by phosphatase PLL5, resulting in the alkalization of the growth medium within 2 mins of systemin treatment. We found the MAP kinase MPK2 to have increased phosphorylation level at its activating TEY-motif at 15 min post-treatment. The predicted interaction of MPK2 with LHA1 was confirmed by in vitro kinase assays, suggesting that the H+-ATPase LHA1 is re-activated by MPK2 later in the systemin response. Our data set provides a resource of proteomic events involved in systemin signaling that will be valuable for further in-depth functional studies in elucidation of systemin signaling cascades.

Development and characterization of bentonite/wGLP systems

Bentonite (Bent) clay is a component of soil with useful properties for enzyme adsorption. In this work, the previously characterized antioxidant superoxide dismutase (SOD) enzyme named wGLP by wheat germin-like protein was immobilized in raw (Bent) and modified bentonites (Bent-DDA). The physicochemical characterizations of both supports were carried out. Successful adsorption of wGLP onto Bent and Bent-DDA occurred on the surface and within the interlayer spacing as was revealed by X-Ray Diffraction (XRD) and Thermogravimetric Analysis (TGA). The release of wGLP at different times and pHs was tested. While wGLP remained almost totally immobilized into Bent-DDA up to 96 h, it was released from Bent to reach nearly 60% after 72 h at pH 7.5 and preserving its SOD activity. Since tomato cell viability under the presence of Bent-wGLP was maintained, Bent-wGLP complexes are potential carriers of antioxidants in tomato cell suspension cultures.

Absorption and Distribution Kinetics of the 13C-Labeled Tomato Carotenoid Phytoene in Healthy Adults

Background: Phytoene is a tomato carotenoid that may contribute to the apparent health benefits of tomato consumption. Although phytoene is a less prominent tomato carotenoid than lycopene, it is a major carotenoid in various human tissues. Phytoene distribution to plasma lipoproteins and tissues differs from lycopene, suggesting the kinetics of phytoene and lycopene differ.Objective: The objective of this study was to characterize the kinetic parameters of phytoene absorption, distribution, and excretion in adults, to better understand why biodistribution of phytoene differs from lycopene.Methods: Four adults (2 males, 2 females) maintained a controlled phytoene diet (1–5 mg/d) for 42 d. On day 14, each consumed 3.2 mg 13C-phytoene, produced using tomato cell suspension culture technology. Blood samples were collected at 0, 1–15, 17, 21, and 24 h and 2, 3, 4, 7, 10, 14, 17, 21, and 28 d after 13C-phytoene consumption. Plasma-unlabeled and plasma-labeled phytoene concentrations were determined using ultra-HPLC–quadrupole time-of-flight-mass spectrometry, and data were fit to a 7-compartment carotenoid kinetic model using WinSAAM 3.0.7 software.Results: Subjects were compliant with a controlled phytoene diet, consuming a mean ± SE of 2.5 ± 0.6 mg/d, resulting in a plasma unlabeled phytoene concentration of 71 ± 14 nmol/L. A maximal plasma 13C-phytoene concentration of 55.6 ± 5.9 nM was achieved 19.8 ± 9.2 h after consumption, and the plasma half-life was 2.3 ± 0.2 d. Compared with previous results for lycopene, phytoene bioavailability was nearly double at 58% ± 19%, the clearance rate from chylomicrons was slower, and the rates of deposition into and utilization by the slow turnover tissue compartment were nearly 3 times greater.Conclusions: Although only differing from lycopene by 4 double bonds, phytoene exhibits markedly different kinetic characteristics in human plasma, providing insight into metabolic processes contributing to phytoene enrichment in plasma and tissues compared with lycopene. This trial was registered at clinicaltrials.gov as NCT01692340.

Modifications of cell wall pectin in tomato cell suspension in response to cadmium and zinc

Retention of metal cations by the cell wall is a common process found in plants in response to stress induced by the presence of trace metals (TMs). In this study conducted on a tomato cell suspension culture, cadmium (Cd) and zinc (Zn) were added to the medium at maximal concentrations of 0.5 and 2 mM, respectively. We showed that around 50 % of Zn or Cd was confined into the cell wall of tomato cells. Besides, their accumulation in the cell wall increased with the exogenous concentration in the culture medium. Characterization of cell wall pectins showed a decrease in the highly methylesterified pectin fraction whereas the weakly methylesterified pectin remained stable in response to Cd. Moreover, a significant increase in the degree of methylesterification was observed in both fractions. This was probably associated to the reduced pectin methylesterase (PME) activity in the treated cells. Furthermore, linked to a reduction of pectin content we showed a reduced expression of the galacturonosyltransferase QUA1 gene whereas PME1 expression remained unchanged. Taking together, these data strongly suggest that pectin biosynthesis and its modification in the cell wall are strongly regulated in response to TM exposure in tomato cells.

A meshfree model for plant tissue deformations during drying

A meshfree model for plant tissue deformations during drying

13C‐phytoene from tomato cell suspension cultures for pharmacokinetic studies in healthy adults (645.15)

Phytoene (C40H64) is a colorless carotenoid found in tomatoes and other plant foods and is enriched in tomato consumers’ plasma and tissues. Yet, little is known about its absorption, distribution, and clearance. To study phytoene pharmacokinetics, phytoene was 13C‐biolabeled using tomato cell suspension cultures grown with uniformly‐labeled 13C‐glucose (30 g/L media) for a 7‐d 13C‐loading and 18‐d 13C‐labeling cycle to yield 290 g cells/L culture. Uniformly labeled 13C‐phytoene was the predominant isotopomer (13C40H56; ~47%), and 蠅85% carbon was 13C across the major isotopomers. 13C‐phytoene was isolated from 2.53 kg of cells by hexane/ethanol/water biphasic extraction and purified by semi‐preparatory C18 HPLC‐PDA. Two wk before test meal consumption, 4 healthy adults (2 females, 2 males, 27.7 ± 2.3 yr, BMI 22 ± 1) began a controlled phytoene diet (1‐5 mg/d) to achieve steady‐state conditions and continued this diet for 4 wk, after 13C‐phytoene consumption. On day 0, subjects consumed 3.2 mg 13C‐phytoene in 10 mL carotenoid‐free olive oil with a low‐carotenoid breakfast. Plasma samples were collected at baseline, hourly from 1‐15 h, at 17, 19, 21, and 24 h, and 2, 3, 4, 7, 10, 14, 17, 21, and 28 d after dosing. Mean dietary phytoene intake was 2.5 ± 0.6 mg/d over 6 wk, with major dietary sources being tomato‐containing products, sweet bell peppers, and winter squash. Plasma 13C‐phytoene concentrations will be measured by HPLC‐MS/MS for pharmacokinetic analyses. Pharmacokinetic investigation of tomato phytochemicals elucidates their biological relevance and promotes development of rational dietary interventions for clinical trial investigations. NIH/NCCAM‐R21AT005166Grant Funding Source: NIH/NCCAM‐R21AT005166

Mechanisms of xylanase-induced nitric oxide and phosphatidic acid production in tomato cells

The second messenger nitric oxide (NO), phosphatidic acid (PA) and reactive oxygen species (ROS) are involved in the plant defense response during plant-pathogen interactions. NO has been shown to participate in PA production in response to the pathogen-associated molecular pattern xylanase in tomato cell suspensions. Defense responses downstream of PA include ROS production. The goal of this work was to study the signaling mechanisms involved in PA production during the defense responses triggered by xylanase and mediated by NO in the suspension-cultured tomato cells. We analyzed the participation of protein kinases, guanylate cyclase and the NO-mediated posttranslational modification S-nitrosylation, by means of pharmacology and biochemistry. We showed that NO, PA and ROS levels are significantly diminished by treatment with the general protein kinase inhibitor staurosporine. This indicates that xylanase-induced protein phosphorylation events might be the important components leading to NO formation, and hence for the downstream regulation of PA and ROS levels. When assayed, a guanylate cyclase inhibitor or a cGMP analog did not alter the PA accumulation. These results suggest that a cGMP-mediated pathway is not involved in xylanase-induced PA formation. Finally, the inhibition of protein S-nitrosylation did not affect NO formation but compromised PA and ROS production. Data collectively indicate that upon xylanase perception, cells activate a protein kinase pathway required for NO formation and that, S-nitrosylation-dependent mechanisms are involved in downstream signaling leading to PA and ROS.

Production of [13C]‐lycopene from high lycopene tomato cell suspension cultures

The goal of this work was to use tomato cell suspension cultures treated with enzyme inhibitors and grown with [13C6]‐glucose as a carbon source to produce labeled carotenoids (CAR) for human metabolism research. To promote CAR accumulation, the high lycopene (LYC) tomato cell suspension line ‘Ailsa Craig’ hp‐1 was grown with carotenogenic enzyme inhibitors norflurazon (NORF), 2‐(4‐chlorophenyl‐thio) triethylamine (CPTA), both herbicides (BOTH), or neither (CONT). Significantly greater combined CAR [phytoene (PE), phytofluene (PF), and LYC] yields were obtained with the NORF, CPTA, and BOTH treatments (5.2 ± 0.7, 3.6 ± 0.4, 4.2 ± 0.4 mg/L, respectively) than CONT (0.3 ± 0.1 mg/L). Different PE, PF, and LYC profiles were obtained where CPTA led to 0.26 ± 0.02, 0.14 ± 0.02, and 3.18 ± 0.45 mg/L; NORF led to 4.96 ± 0.68, 0.20 ± 0.06, and 0.06 ± 0.02 mg/L; BOTH led to 2.98 ± 0.14, 0.18 ± 0.05, and 1.05 ± 0.36 mg/L; CONT led to 0.18 ± 0.09, 0.02 ± 0.00, and 0.06 ± 0.01 mg/L, respectively. To biolabel LYC the hp‐1 cell line was grown in medium containing 30 g/L [13C6]‐glucose and CPTA. LYC, PE, and PF yields from labeled cultures were 2.5 ± 0.1, 0.3 ± 0.0, 0.1 ± 0.0 mg/L, respectively. Mass isotopomer distribution analysis for LYC [13C]‐enrichment from a pilot labeling run was performed using LC‐MS/MS, and 82% of the LYC molecules had at least 34 of 40 carbons labeled, and the greatest percent (17%) was uniformly labeled. (NIH/NCI CA 112649‐01A1)

Phosphatidic acid formation is required for extracellular ATP‐mediated nitric oxide production in suspension‐cultured tomato cells

*In animals and plants, extracellular ATP exerts its effects by regulating the second messengers Ca(2+), nitric oxide (NO) and reactive oxygen species (ROS). In animals, phospholipid-derived molecules, such as diacylglycerol, phosphatidic acid (PA) and inositol phosphates, have been associated with the extracellular ATP signaling pathway. The involvement of phospholipids in extracellular ATP signaling in plants, as it is established in animals, is unknown. *In vivo phospholipid signaling upon extracellular ATP treatment was studied in (32)P(i)-labeled suspension-cultured tomato (Solanum lycopersicum) cells. *Here, we report that, in suspension-cultured tomato cells, extracellular ATP induces the formation of the signaling lipid phosphatidic acid. Exogenous ATP at doses of 0.1 and 1 mM induce the formation of phosphatidic acid within minutes. Studies on the enzymatic sources of phosphatidic acid revealed the participation of both phospholipase D and C in concerted action with diacylglycerol kinase. *Our results suggest that extracellular ATP-mediated nitric oxide production is downstream of phospholipase C/diacylglycerol kinase activation.

Reassessing the role of phospholipase D in theArabidopsiswounding response

Plants respond to wounding by means of a multitude of reactions, with the purpose of stifling herbivore assault. Phospholipase D (PLD) has previously been implicated in the wounding response. Arabidopsis (Arabidopsis thaliana) AtPLDalpha1 has been proposed to be activated in intact cells, and the phosphatidic acid (PA) it produces to serve as a precursor for jasmonic acid (JA) synthesis and to be required for wounding-induced gene expression. Independently, PLD activity has been reported to have a bearing on wounding-induced MAPK activation. However, which PLD isoforms are activated, where this activity takes place (in the wounded or non-wounded cells) and what exactly the consequences are is a question that has not been comprehensively addressed. Here, we show that PLD activity during the wounding response is restricted to the ruptured cells using (32)P(i)-labelled phospholipid analyses of Arabidopsis pld knock-out mutants and PLD-silenced tomato cell-suspension cultures. pldalpha1 knock-out lines have reduced wounding-induced PA production, and the remainder is completely eliminated in a pldalpha1/delta double knock-out line. Surprisingly, wounding-induced protein kinase activation, AtLOX2 gene expression and JA biosynthesis were not affected in these knock-out lines. Moreover, larvae of the Cabbage White butterfly (Pieris rapae) grew equally well on wild-type and the pld knock-out mutants.

Cadmium-Induced Programmed Cell Death Signaling in Tomato Suspension Cells

ABSTRACTHere we present a summary of our study on cadmium-induced cell death signaling in a model system of suspension-cultured tomato cells. Exposure of the cells to CdSO4 induced typical for PCD (cytoplasm shrinkage and nuclear condensation) morphological changes of the dead cells. Ethylene and hydrogen peroxide were established to increase in response to Cd stress. By inhibitory studies we have demonstrated that CdSO4 induces cell death in tomato suspension cells that involve plant caspase-like proteases, indicating that Cd-triggered cell death is a form of programmed cell death. It is also shown that serine peptidases, oxidative stress, lipid signaling and ethylene are players in the Cd-induced cell death signaling. Cd-induced cell death in tomato suspension cells was categorised as non-lysosomal type.

Optimization of Lycopene Extraction from Tomato Cell Suspension Culture by Response Surface Methodology

Radioisotope-labeled lycopene is an important tool for biomedical research but currently is not commercially available. A tomato cell suspension culture system for the production of radioisotope-labeled lycopene was previously developed in our laboratory. In the current study, the goal was to optimize the lycopene extraction efficiency from tomato cell cultures for preparatory high-performance liquid chromatography (HPLC) separation. We employed response surface methodology (RSM), which combines fractional factorial design and a second-degree polynomial model. Tomato cells were homogenized with ethanol, saponified by KOH, and extracted with hexane, and the lycopene content was analyzed by HPLC-PDA. We varied five factors at five levels: ethanol volume (1.33-4 mL/g); homogenization period (0-40 s/g); saturated KOH solution volume (0-0.67 mL/g); hexane volume (1.67-3 mL/g); and vortex period (5-25 s/g). Ridge analysis by SAS suggested that the optimal extraction procedure to extract 1 g of tomato cells was at 1.56 mL of ethanol, 28 s homogenization, 0.29 mL of KOH, 2.49 mL of hexane, and 17.5 s vortex. These optimal conditions predicted by RSM were confirmed to enhance lycopene yield from standardized tomato cell cultures by more than 3-fold.

Optimization of Lycopene Extraction from Tomato Cell Culture

For biomedical research, radioisotope‐labeled lycopene is important but not commercially available. A tomato cell suspension culture system for the production of radioisotope‐labeled lycopene was previously developed in our laboratory. In this study, our aim was to improve the lycopene extraction efficiency from the tomato cell culture. We employed the response surface methodology (RSM) which combines fractional factorial design and a second‐degree polynomial model. Tomato cells were homogenized with ethanol, saponified by KOH, extracted by hexane, and the lycopene content was analyzed by HPLC. We varied five factors with five levels: ethanol volume (4–12 mL); homogenization period (0–120 second); saturated KOH solution volume (0–2 mL); hexane volume (5–9 mL); and vortex period (15–75 second). RIDGE analysis by SAS suggested that the optimal extraction procedure employed 9.5 mL ethanol, 75 seconds homogenization, 0.94 mL KOH, 6.4 mL hexane, and 36 seconds vortex to yield lycopene 13.9 nmol lycopene /g from 3 g tomato cells. By repeating this predicted condition, the improvement of extraction yield was confirmed. The production of radioisotope‐labeled lycopene was more efficient and economical with improvement of the extraction procedure by RSM.

Production of distinct carotenoid mixtures from tomato cell suspension cultures varies by herbicide treatment and variety used.

Tomato product consumption has been correlated with decreased chronic disease risk. Radiolabeled forms of tomato carotenoids are essential for tracking bioavailability, biodistribution, and metabolism in human and animal models; however, many of these compounds are not commercially available. In order to target specific compounds and efficiently produce mixtures of tomato carotenoids [phytoene (PE), phytofluene (PF), and lycopene (LYC)] for future radiolabeling, different herbicide treatments and tomato cell lines were investigated. Three herbicide treatments [norflurazon (NORF), 2‐(4‐chlorophenyl‐thio) triethylamine (CPTA), or NORF & CPTA (COMBO)] were investigated with ‘VFNT Cherry’. CPTA treatment yielded primarily LYC (2.49 mg/L culture) followed by PE & PF (0.30, 0.32 mg/L). NORF treatment yielded the greatest PE (1.00 mg/L) followed by PF (0.10 mg/L) and traces of LYC. COMBO treatment led to the best‐mixed yields of PE, PF, and LYC (1.09, 0.28, 1.45 mg/L). Five different cell lines (‘VFNT Cherry’, ‘Ailsa Craig’ hp‐1, ‘Ailsa Craig’ WT, ‘Manapal’ hp‐2, and ‘Manapal’ WT) were treated with CPTA and evaluated, and ‘VFNT Cherry’ and the ‘Ailsa Craig’ hp‐1 mutant accumulated the greatest LYC. Selected varieties and herbicide treatments will be applied for radiolabeled carotenoid generation for use in future animal and human trials. (Supported by NIH/NCI CA 112649‐01A1)

Host selectivity of a 40kDa phytotoxic extract fromColletotrichum gloeosporioides(Phyllachoraceae) on yamDioscorea alata(Dioscoreace)

Isolates of the fungal pathogen Deuteromycota, Melanconiales, Colletotrichum gloeosporioides (Penz and Sacc.) from yam, Dioscoreales, Dioscoreacceae, Dioscorea alata cultivar White Lisbon, and one each from Mangifera indica L (mango), Anthurium andreanum (Schott) (anthurium) and Citrus paradisi (grapefruit) were used for precipitation of phytotoxic secondary metabolites. These phytotoxic extracts decreased cell viability of yam, tomato, pepper and tobacco cell suspension cultures. Protein and carbohydrate fractions of the extract co-eluted during gel filtration chromatography and were present in amounts ranging from 26–46 μgmL-1 and 0.24–0.62 μgmL-1 respectively per gram of phytotoxin. Biochemical analyses of the phytotoxic extracts showed they contained mainly two sugars, mannose, and galactose which were both closely linked to a protein component containing large amounts of cysteine and glycine. This latter protein component was essential for disease induction in yam and is part of a relatively mid-range glycoprotein complex containing both N and O-glycosidic bonds, approximately 40kDa in molecular mass.

Nitric Oxide Is Critical for Inducing Phosphatidic Acid Accumulation in Xylanase-elicited Tomato Cells

Nitric Oxide (NO) is a second messenger related to development and (a)biotic stress responses in plants. We have studied the role of NO in signaling during plant defense responses upon xylanase elicitation. Treatment of tomato cell cultures with the fungal elicitor xylanase resulted in a rapid and dose-dependent NO accumulation. We have demonstrated that NO is required for the production of the lipid second messenger phosphatidic acid (PA) via the activation of the phospholipase C (PLC) and diacylglycerol kinase (DGK) pathway. Defense-related responses downstream of PA were studied. PA and, correspondingly, xylanase were shown to induce reactive oxygen species production. Scavenging of NO or inhibition of either the PLC or the DGK enzyme diminished xylanase-induced reactive oxygen species production. Xylanase-induced PLDbeta1 and PR1 mRNA levels decreased when NO or PA production were compromised. Finally, we have shown that NO and PA are involved in the induction of cell death by xylanase. Treatment with NO scavenger cPTIO, PLC inhibitor U73122, or DGK inhibitor R59022 diminished xylanase-induced cell death. On the basis of biochemical and pharmacological experimental results, we have shown that PLC/DGK-derived PA represents a novel downstream component of NO signaling cascade during plant defense.

Selection, characterization, and herbicidal enzymatic inhibition of high carotenoid producing tomato cell suspension cultures

Selection, characterization, and herbicidal enzymatic inhibition of high carotenoid producing tomato cell suspension cultures