Bioprocessing of camptothecin from Alternaria brassicicola, an endophyte of Catharanthus roseus, with a strong antiproliferative activity and inhibition to Topoisomerases.

Suppression of fungal camptothecin (CPT) biosynthesis with the preservation and successive subculturing is the challenge that impedes fungi from the industrial application, so, screening for a novel fungal isolate with a conceivable stable producing potency of CPT was the main objective of this work. Catharanthus roseus with diverse contents of bioactive metabolites could have a plethora of novel endophytes with unique metabolic properties. Among the endophytes of C. roseus, Alternaria brassicicola EFBL-NV OR131587.1 was the highest CPT producer (96.5 µg/L). The structural identity of the putative CPT was verified by HPLC, FTIR, HNMR and LC-MS/MS, with a molecular mass 349 m/z, and molecular fragmentation patterns that typically identical to the authentic one. The purified A. brassicicola CPT has a strong antiproliferative activity towards UO-31 (0.75 µM) and MCF7 (3.2 µM), with selectivity index 30.8, and 7.1, respectively, in addition to resilient activity to inhibit Topo II (IC50 value 0.26 nM) than Topo 1 (IC50 value 3.2 nM). The purified CPT combat the wound healing of UO-31 cells by ~ 52%, stops their matrix formation, cell migration and metastasis. The purified CPT arrest the cellular division of the UO-31 at the S-phase, and inducing their cellular apoptosis by ~ 20.4 folds, compared to the control cells. Upon bioprocessing with the surface response methodology, the CPT yield by A. brassicicola was improved by ~ 3.3 folds, compared to control. The metabolic potency of synthesis of CPT by A. brassicicola was attenuated with the fungal storage and subculturing, losing ~ 50% of their CPT productivity by the 6th month of storage and 6th generation. Practically, the CPT productivity of the attenuated A. brassicicola was restored by addition of 1% surface sterilized leaves of C. roseus, ensuring the eliciting of cryptic gene cluster of A. brassicicola CPT via the plant microbiome-A. brassicicola interactions. So, for the first time, a novel endophytic isolate A. brassicicola, from C. roseus, was explored to have a relatively stable CPT biosynthetic machinery, with an affordable feasibility to restore their CPT productivity using C. roseus microbiome, in addition to the unique affinity of the extracted CPT to inhibit Topoisomerase I and II.

Camptothecin bioprocessing from Aspergillus terreus, an endophyte of Catharanthus roseus: antiproliferative activity, topoisomerase inhibition and cell cycle analysis.

Attenuation of camptothecin (CPT) productivity by fungi with preservation and subculturing is the challenge that halts fungi to be an industrial platform of CPT production. Thus, screening for novel endophytic fungal isolates with metabolic stability for CPT production was the objective. Catharanthus roseus is one of the medicinal plants with diverse bioactive metabolites that could have a plethora of novel endophytes with unique metabolites. Among the endophytes of C. roseus, Aspergillus terreus EFBL-NV OR131583.1 had the most CPT producing potency (90.2 µg/l), the chemical identity of the putative CPT was verified by HPLC, FT-IR, NMR and LC-MS/MS. The putative A. terreus CPT had the same molecular mass (349 m/z), and molecular fragmentation patterns of the authentic one, as revealed from the MS/MS analyses. The purified CPT had a strong activity against MCF7 (5.27 µM) and UO-31 (2.2 µM), with a potential inhibition to Topo II (IC50 value 0.52 nM) than Topo 1 (IC50 value 6.9 nM). The CPT displayed a high wound healing activity to UO-31 cells, stopping their metastasis, matrix formation and cell immigration. The purified CPT had a potential inducing activity to the cellular apoptosis of UO-31 by ~ 17 folds, as well as, arresting their cellular division at the S-phase, compared to the control cells. Upon Plackett-Burman design, the yield of CPT by A. terreus was increased by ~ 2.6 folds, compared to control. The yield of CPT by A. terreus was sequentially suppressed with the fungal storage and subculturing, losing ~ 50% of their CPT productivity by 3rd month and 5th generation. However, the productivity of the attenuated A. terreus culture was completely restored by adding 1% surface sterilized leaves of C. roseus, and the CPT yield was increased over-the-first culture by ~ 3.2 folds (315.2 µg/l). The restoring of CPT productivity of A. terreus in response to indigenous microbiome of C. roseus, ensures the A. terreus-microbiome interactions, releasing a chemical signal that triggers the CPT productivity of A. terreus. This is the first reports exploring the potency of A. terreus, endophyte of C. roseus" to be a platform for industrial production of CPT, with an affordable sustainability with addition of C. roseus microbiome.

Exploring genotypic variability and interrelationships among growth, yield, and quality characteristics in diverse tomato genotypes.

Tomato is the most consumed vegetable crop worldwide, with excellent beneficial health properties and high content of vitamins, minerals, carotenoids, total antioxidants, and phenolic compounds. Hence, improving its genotypes is crucial to sustain its production and ensure food security, principally under the fast-growing worldwide population and abrupt global climate change. The present study aimed to explore the genotypic variability associated with specific characteristics in twenty-five diverse tomato genotypes. In addition, the relationships between growth, yield, and quality traits using both univariate (correlation coefficient, path analysis) and multivariate (principal component, principal coordinates, canonical variate) analysis methods were explored. The results indicated that the evaluated genotypes possessed highly significant variation. This is appropriate for future hybridization through tomato breeding programs. All evaluated genotypes demonstrated considerable potential to develop strong hybrid vigour for growth, yield, and quality characteristics. In particular, the genotypes LS009, LS011, and LS014 could be considered promising, high-yielding, and resistant to yellow leaf curl virus infestation (YLCV) disease parents for future breeding schemes. The number of fruits per plant, fruit diameter, and fruit weight proved strong positive relationships with fruit yield. Accordingly, these characteristics demonstrate their importance in improving fruit yield and could be exploited as indirect criteria for selecting high-yielding tomato genotypes through breeding programs.

Polyamines mitigate the destructive impacts of salinity stress by enhancing photosynthetic capacity, antioxidant defense system and upregulation of calvin cycle-related genes in rapeseed (Brassica napus L.).

Salinity is widespread environmental stress that poses great obstacles to rapeseed development and growth. Polyamines are key plant growth regulators that play a pivotal role in regulating salt tolerance. Rapeseed (Brassica napus L.) seedlings were treated by spermine (Spm) and spermidine (Spd) versus untreated control under salt stress conditions. It was detected that the Spd-treated plants had significantly elevated chlorophyll and proline content and maintained higher photosystem II (PSII) activity than those treated with Spm as well as untreated control under salt-stressed conditions. Similarly, Spd alleviated the devastating effects of NaCl stress on CO2 assimilation and significantly elevated Rubisco activity (ribulose 1,5-bisphosphate carboxylase/oxygenase). The application of Spd also enhanced the activities of different antioxidant enzymes under NaCl stress. It modulated their respective transcription levels, including ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and dehydroascorbate reductase (DHAR). In addition, exogenously sprayed Spd enhanced the polyamine pathway as observed by upregulated transcription of polyamine oxidase (PAO) and diamine oxidase (DAO). The Spd application enhanced expressions of Calvin cycle enzyme related genes such as Rubisco small subunit, Rubisco large subunit, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglyceric acid kinase (PGK), triose-3-phosphate isomerase (TPI), fructose-1,6-bisphosphate aldolase (FBA), sedoheptulose-1,7-bisphosphatase (SBPase), and fructose-1,6-bisphosphate phosphatase (FBPase). Consequently, this study demonstrates that exogenous application of Spd has a valuable role in regulating antioxidant enzyme activity, polyamine pathway, and Calvin cycle enzyme-related genes to alleviate salt stress damage in the plants.

Assessing the Response of Diverse Sesame Genotypes to Waterlogging Durations at Different Plant Growth Stages.

Sesame is sensitive to waterlogging, and its growth is devastatingly impacted under excess moisture conditions. Thus, waterlogging tolerance is crucial to alleviate yield constraints, particularly under expected climate change. In this study, 119 diverse sesame genotypes were screened for their tolerance to 12, 24, 48, and 72 h of waterlogging relative to non-waterlogged conditions. All plants died under 72 h of waterlogging, while 13.45%, 31.93%, and 45.38% of genotypes survived at 48, 24, and 12 h, respectively. Based on the seedling parameters and waterlogging tolerance coefficients, genotypes BD-7008 and BD-6985 exhibited the highest tolerance to waterlogging, while BD-6996 and JP-01811 were the most sensitive ones. The responses of these four genotypes to waterlogged conditions were assessed at different plant growth stages-30, 40, and 50 days after sowing (DAS)-versus normal conditions. Waterlogging, particularly when it occurred within 30 DAS, destructively affected the physiological and morphological characteristics, which was reflected in the growth and yield attributes. Genotype BD-7008, followed by BD-6985, exhibited the highest chlorophyll and proline contents as well as enzymatic antioxidant activities, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). These biochemical and physiological adjustments ameliorated the adverse effects of waterlogging, resulting in higher yields for both genotypes. Conversely, JP-01811 presented the lowest chlorophyll and proline contents as well as enzymatic antioxidant activities, resulting in the poorest growth and seed yield.

Melatonin Regulatory Mechanisms and Phylogenetic Analyses of Melatonin Biosynthesis Related Genes Extracted from Peanut under Salinity Stress.

Melatonin improves the tolerance of plants to various environmental stresses by protecting plant cells against oxidative stress damage. The objective of the current study was to determine whether exogenous melatonin (MT) treatments could help protecting peanut (Arachis hypogaea) seedlings against salinity stress. This was achieved by investigating enzymatic and non-enzymatic antioxidant systems and the expression of melatonin biosynthesis related genes in response to salinity stress with or without exogenous MT. The results showed a significant increase in the concentrations of reactive oxygen species (ROS) in peanut seedlings under salinity stress. The exogenous application of melatonin decreased the levels of ROS through the activation of antioxidant enzymes in peanut seedlings under salinity stress. Transcription levels of melatonin biosynthesis related genes such as N-acetylserotonin methyltransferase (ASMT1, ASMT2, ASMT3), tryptophan decarboxylase (TDC), and tryptamine 5-hydroxylase (T5H) were up-regulated with a 150 µM melatonin treatment under salinity stress. The results indicated that melatonin regulated the redox homeostasis by its ability to induce either enzymatic or non-enzymatic antioxidant systems. In addition, phylogenetic analysis of melatonin biosynthesis genes (ASMT1, ASMT2, ASMT3, TDC, T5H) were performed on a total of 56 sequences belonging to various plant species including five new sequences extracted from Arachis hypogaea (A. hypogaea). This was based on pairwise comparison among aligned nucleotides and predicted amino acids as well as on substitution rates, and phylogenetic inference. The analyzed sequences were heterogeneous and the A. hypogaea accessions were primarily closest to those of Manihot esculenta, but this needs further clarification.

Stimulating antioxidant defenses, antioxidant gene expression, and salt tolerance in Pisum sativum seedling by pretreatment using licorice root extract (LRE) as an organic biostimulant.

Plant extracts have recently been used as exogenous adjuvants to strengthen the endogenous plant defense systems when they grow under different environmental stresses, including salinity. The study aimed at determining the effects of seed soaking using licorice root extract (LRE) on photosynthesis and antioxidant defense systems, including transcript levels of enzyme-encoding genes in pea seedling grown under 150 mM NaCl-salinity. Salt stress reduced seedling growth, photosynthesis attributes, and K+ content, and increased oxidative stress (O2•‒ and H2O2, and MDA), Na+, and Cl-, along with an increase in antioxidative defense activities compared to control. However, LRE pretreatment enhanced seedling growth, photosynthetic attributes (chlorophylls, carotenoids, Fv/Fm, Pn, Tr, and gs), ascorbate and glutathione and their redox states, proline, soluble sugars, α-TOC, and enzyme activities compared to stressed control. LRE pretreatment also upregulated transcript levels of CAT-, SOD-, APX-, GR-, DHAR-, and PrxQ-encoding genes in salt-stressed seedlings, decreasing oxidative stress and Na+ and Cl- contents and increasing K+ content and K+/Na+ ratio.

The Impact of Drought Stress on Antioxidant Responses and Accumulation of Flavonolignans in Milk Thistle (Silybum marianum (L.) Gaertn).

Biosynthesis and accumulation of flavonolignans in plants are influenced by different environmental conditions. Biosynthesis and accumulation of silymarin in milk thistle (Silybum marianum L.) were studied under drought stress with respect to the antioxidant defense system at the physiological and gene expression level. The results revealed a reduction in leaf chlorophyll, ascorbic acid, and glutathione contents. In contrast, H2O2, proline, and antioxidative enzyme activities, such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR), were increased. These results confirmed that milk thistle undergoes oxidative stress under drought stress. Furthermore, transcription levels of APX, SOD, CAT, 1-Cys-Prx, and PrxQ were significantly increased in milk thistle under drought stress. Overall this suggests that protection against reactive oxygen species and peroxidation reactions in milk thistle are provided by enzymatic and non-enzymatic antioxidants. Flavonolignans from milk thistle seeds after different drought treatments were quantified by high-performance liquid chromatography (HPLC) and showed that severe drought stress enhanced the accumulation of silymarin and its components compared with seeds from the control (100% water capacity). Silybin is the major silymarin component and the most bioactive ingredient of the milk thistle extract. Silybin accumulation was the highest among all silymarin components in seeds obtained from drought-stressed plants. The expression of the chalcone synthase (CHS) genes (CHS1, CHS2, and CHS3), which are associated with the silybin biosynthetic pathway, was also increased during drought stress. These results indicated that milk thistle exhibits tolerance to drought stress and that seed derived from severe drought-stressed plants had higher levels of silymarin.

Enhancing antioxidant systems by exogenous spermine and spermidine in wheat (Triticum aestivum) seedlings exposed to salt stress.

Plants have evolved complex mechanisms to mitigate osmotic and ionic stress caused by high salinity. The effect of exogenous spermine (Spm) and spermidine (Spd) on defence responses of wheat seedlings under NaCl stress was investigated by measuring antioxidant enzyme activities and the transcript expression of corresponding genes. Exogenous Spm and Spd decreased the level of malondialdehyde, increased chlorophyll and proline contents, and modulated PSII activity in wheat seedlings under salt stress. Spermidine alleviated negative effects on CO2 assimilation induced by salt stress in addition to significantly increasing the activity and content of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). It appears Spd conferred salinity tolerance in wheat seedlings by enhancing photosynthetic capacity through regulation of gene expression and the activity of key CO2 assimilation enzymes. Exogenous Spm regulated activities of different antioxidant enzymes (catalase, glutathione reductase, dehydroascorbate reductase, ascorbate peroxidase, and superoxide dismutase) and efficiently modulate their transcription levels in wheat seedlings under salt stress. It is likely that Spm plays a key role in alleviating oxidative damage of salt stress by adjusting antioxidant enzyme activities in plants. In addition, exogenous Spd increased transcript level of spermine synthase under salt stress. Salinity stress also caused an increase in transcript levels of diamine oxidase (DAO) and polyamine oxidase (PAO). Exogenous Spd application resulted in a marked increase in free Spd and Spm contents under saline conditions. These results show that exogenous Spd and Spm effectively upregulated transcriptional levels of antioxidant enzyme genes and improved the defence response of plants under salt stress.

Lectin approaches for glycoproteomics in FDA-approved cancer biomarkers.

The nine FDA-approved protein biomarkers for the diagnosis and management of cancer are approaching maturity, but their different glycosylation compositions relevant to early diagnosis still remain practically unexplored at the sub-glycoproteome scale. Lectins generally exhibit strong binding to specific sub-glycoproteome components and this property has been quite poorly addressed as the basis for the early diagnosis methods. Here, we discuss some glycoproteome issues that make tackling the glycoproteome particularly challenging in the cancer biomarkers field and include a brief view for next generation technologies.

Preferential lectin binding of cancer cells upon sialic acid treatment under nutrient deprivation.

The terminal monosaccharide of glycoconjugates on a eukaryotic cell surface is typically a sialic acid (Neu5Ac). Increased sialylation usually indicates progression and poor prognosis of most carcinomas. Here, we utilize two human mammary epithelial cell lines, HB4A (breast normal cells) and T47D (breast cancer cells), as a model system to demonstrate differential surface glycans when treated with sialic acid under nutrient deprivation. Under a starved condition, sialic acid treatment of both cells resulted in increased activities of α2→3/6 sialyltransferases as demonstrated by solid phase assay using lectin binding. However, a very strong Maackia amurensis agglutinin I (MAL-I) staining on the membrane of sialic acid-treated T47D cells was observed, indicating an increase of Neu5Acα2→3Gal on the cell surface. To our knowledge, this is a first report showing the utility of lectins, particularly MAL-I, as a means to discriminate between normal and cancer cells after sialic acid treatment under nutrient deprivation. This method is sensitive and allows selective detection of glycan sialylation on a cancer cell surface.