The Effect of Cold Stress on the Root-Specific Lipidome of Two Wheat Varieties with Contrasting Cold Tolerance.

Complex glycerolipidome analysis of wheat upon low temperature stress has been reported for above-ground tissues only. There are no reports on the effects of cold stress on the root lipidome nor on tissue-specific responses of cold stress wheat roots. This study aims to investigate the changes of lipid profiles in the different developmental zones of the seedling roots of two wheat varieties with contrasting cold tolerance exposed to chilling and freezing temperatures. We analyzed 273 lipid species derived from 21 lipid classes using a targeted profiling approach based on MS/MS data acquired from schedule parallel reaction monitoring assays. For both the tolerant Young and sensitive Wyalkatchem species, cold stress increased the phosphatidylcholine and phosphatidylethanolamine compositions, but decreased the monohexosyl ceramide compositions in the root zones. We show that the difference between the two varieties with contrasting cold tolerance could be attributed to the change in the individual lipid species, rather than the fluctuation of the whole lipid classes. The outcomes gained from this study may advance our understanding of the mechanisms of wheat adaptation to cold and contribute to wheat breeding for the improvement of cold-tolerance.

Stem Hexane Extract of Strobilanthes crispus Induces Apoptosis in Triple-Negative Breast Cancer Cell Line.

Strobilanthes crispus is known to possess multiple health beneficial effects and reported to be traditionally used as medicine in several countries. This study was to investigate the anti-proliferative effects of S. crispus leaves and stem extracts on MDA-MB-231 by examining their effects on apoptosis pathway. The chemical compounds were extracted from leaves and stems using methanol followed by solvent partitioning. Two extracts were found to prevent MDA-MB-231 cell growth at the IC50 of 45 µg/mL and 60 µg/mL, respectively, for leaf water (LW) and stem hexane (SH) extracts. Results showed that SH extract induces apoptosis by suppressing the protein expression of BCL-2 while the expression of pro-apoptotic proteins such as BAX and caspase nine were unchanged. Decrease of cyclin A2 in SH-treated cells suggested this effect was associated with the dysregulation of cell cycle. However, LW extract showed no effects on apoptosis and cell cycle arrest in the treated cells. Taken together, our results showed SH extract of S. crispus exhibiting their anti-proliferative activities by modulating apoptosis and cell cycle, but the underlying mechanisms exerted by LW extract requires further investigation.

Spatially Enriched Paralog Rearrangements Argue Functionally Diverse Ribosomes Arise during Cold Acclimation in Arabidopsis.

Ribosome biogenesis is essential for plants to successfully acclimate to low temperature. Without dedicated steps supervising the 60S large subunits (LSUs) maturation in the cytosol, e.g., Rei-like (REIL) factors, plants fail to accumulate dry weight and fail to grow at suboptimal low temperatures. Around REIL, the final 60S cytosolic maturation steps include proofreading and assembly of functional ribosomal centers such as the polypeptide exit tunnel and the P-Stalk, respectively. In consequence, these ribosomal substructures and their assembly, especially during low temperatures, might be changed and provoke the need for dedicated quality controls. To test this, we blocked ribosome maturation during cold acclimation using two independent reil double mutant genotypes and tested changes in their ribosomal proteomes. Additionally, we normalized our mutant datasets using as a blank the cold responsiveness of a wild-type Arabidopsis genotype. This allowed us to neglect any reil-specific effects that may happen due to the presence or absence of the factor during LSU cytosolic maturation, thus allowing us to test for cold-induced changes that happen in the early nucleolar biogenesis. As a result, we report that cold acclimation triggers a reprogramming in the structural ribosomal proteome. The reprogramming alters the abundance of specific RP families and/or paralogs in non-translational LSU and translational polysome fractions, a phenomenon known as substoichiometry. Next, we tested whether the cold-substoichiometry was spatially confined to specific regions of the complex. In terms of RP proteoforms, we report that remodeling of ribosomes after a cold stimulus is significantly constrained to the polypeptide exit tunnel (PET), i.e., REIL factor binding and functional site. In terms of RP transcripts, cold acclimation induces changes in RP families or paralogs that are significantly constrained to the P-Stalk and the ribosomal head. The three modulated substructures represent possible targets of mechanisms that may constrain translation by controlled ribosome heterogeneity. We propose that non-random ribosome heterogeneity controlled by specialized biogenesis mechanisms may contribute to a preferential or ultimately even rigorous selection of transcripts needed for rapid proteome shifts and successful acclimation.

Arabidopsis REI-LIKE proteins activate ribosome biogenesis during cold acclimation.

Arabidopsis REIL proteins are cytosolic ribosomal 60S-biogenesis factors. After shift to 10 °C, reil mutants deplete and slowly replenish non-translating eukaryotic ribosome complexes of root tissue, while controlling the balance of non-translating 40S- and 60S-subunits. Reil mutations respond by hyper-accumulation of non-translating subunits at steady-state temperature; after cold-shift, a KCl-sensitive 80S sub-fraction remains depleted. We infer that Arabidopsis may buffer fluctuating translation by pre-existing non-translating ribosomes before de novo synthesis meets temperature-induced demands. Reil1 reil2 double mutants accumulate 43S-preinitiation and pre-60S-maturation complexes and alter paralog composition of ribosomal proteins in non-translating complexes. With few exceptions, e.g. RPL3B and RPL24C, these changes are not under transcriptional control. Our study suggests requirement of de novo synthesis of eukaryotic ribosomes for long-term cold acclimation, feedback control of NUC2 and eIF3C2 transcription and links new proteins, AT1G03250, AT5G60530, to plant ribosome biogenesis. We propose that Arabidopsis requires biosynthesis of specialized ribosomes for cold acclimation.

Phenotyping the Chilling and Freezing Responses of Young Microspore Stage Wheat Spikes Using Targeted Metabolome and Lipidome Profiling.

Chilling and frost conditions impose major yield restraints to wheat crops in Australia and other temperate climate regions. Unpredictability and variability of field frost events are major impediments for cold tolerance breeding. Metabolome and lipidome profiling were used to compare the cold response in spikes of cold-tolerant Young and sensitive variety Wyalkatchem at the young microspore (YM) stage of pollen development. We aimed to identify metabolite markers that can reliably distinguish cold-tolerant and sensitive wheat varieties for future cold-tolerance phenotyping applications. We scored changes in spike metabolites and lipids for both varieties during cold acclimation after initial and prolonged exposure to combined chilling and freezing cycles (1 and 4 days, respectively) using controlled environment conditions. The two contrasting wheat varieties showed qualitative and quantitative differences in primary metabolites involved in osmoprotection, but differences in lipid accumulation most distinctively separated the cold response of the two wheat lines. These results resemble what we previously observed in flag leaves of the same two wheat varieties. The fact that this response occurs in tissue types with very different functions indicates that chilling and freezing tolerance in these wheat lines is associated with re-modelling of membrane lipid composition to maintain membrane fluidity.

Morphological and metabolic responses to salt stress of rice (Oryza sativa L.) cultivars which differ in salinity tolerance.

Salinization is one of the most important abiotic stressors for crop growth and productivity. Rice (Oryza sativa L.), as the major food source around the world, is very sensitive to salt, especially at seedling stage. In order to examine how salt stress influences the metabolism of rice, we compared the levels of a range of sugars and organic acids in three rice cultivars with different tolerance under salt stress over time. According to the morphological result, the shoot length and root fresh weight were only affected by salinity in the salt sensitive cultivar (Nipponbare). The responses of metabolites to salinity were time-, tissue- and cultivar-dependent. Shikimate and quinate, involved in the shikimate pathway, were dramatically decreased in the leaves of all three cultivars, which was regarded as a response to salinity. Many sugars in the leaves of the salt tolerant cultivar (Dendang and Fatmawati) showed earlier increases to salt stress compared to Nipponbare leaves. Moreover, only in the leaves of tolerant cultivars (Dendang and Fatimawati), malate was significantly decreased while sucrose was significantly increased. In Dendang roots, mannitol levels were significantly higher than in Nipponbare roots after 14 days of salt treatment, which may be attributed to its higher salt tolerance. It is proposed that these responses in the more tolerant cultivars are involved in their resistance to high salt stress which may lay the foundation for breeding tolerant rice cultivars.

Phenotyping reproductive stage chilling and frost tolerance in wheat using targeted metabolome and lipidome profiling.

INTRODUCTION: Frost events lead to A$360 million of yield losses annually to the Australian wheat industry, making improvement of chilling and frost tolerance an important trait for breeding. OBJECTIVES: This study aimed to use metabolomics and lipidomics to explore genetic variation in acclimation potential to chilling and to identify metabolite markers for chilling tolerance in wheat. METHODS: We established a controlled environment screening assay that is able to reproduce field rankings of wheat germplasm for chilling and frost tolerance. This assay, together with targeted metabolomics and lipidomics approaches, were used to compare metabolite and lipid levels in flag leaves of two wheat varieties with contrasting chilling tolerance. RESULTS: The sensitive variety Wyalkatchem showed a strong reduction in amino acids after the first cold night, followed by accumulation of osmolytes such as fructose, glucose, putrescine and shikimate over a 4-day period. Accumulation of osmolytes is indicative of acclimation to water stress in Wyalkatchem. This response was not observed for tolerant variety Young. The two varieties also displayed significant differences in lipid accumulation. Variation in two lipid clusters, resulted in a higher unsaturated to saturated lipid ratio in Young after 4 days cold treatment and the lipids PC(34:0), PC(34:1), PC(35:1), PC(38:3), and PI(36:4) were the main contributors to the unsaturated to saturated ratio change. This indicates that Young may have superior ability to maintain membrane fluidity following cold exposure, thereby avoiding membrane damage and water stress observed for Wyalkatchem. CONCLUSION: Our study suggests that metabolomics and lipidomics markers could be used as an alternative phenotyping method to discriminate wheat varieties with differences in cold acclimation.

Phyla nodiflora L. Extracts Induce Apoptosis and Cell Cycle Arrest in Human Breast Cancer Cell Line, MCF-7.

Phyla nodiflora L. has been used as medicinal remedies for various ailments due to its antioxidant, anti-inflammatory, anti-bacterial, anti-tumor activity. Previously, we found that the plant extracts induced DNA fragmentation in MCF-7. This study was to investigate the modes of action of P. nodiflora in inhibiting breast cancer cells using leaf ethyl acetate (EA leaf), stem ethyl acetate (EA stem) and stem methanol (Met stem) extracts. The MTT assay showed that the anti-proliferative effects of P. nodiflora extracts were selective towards MCF-7 with a minimal effect on MCF10A. Morphological changes such as cell shrinkage and nuclear condensation were observed in treated cells. We found that induction of apoptosis by EA leaf and EA stem was mitochondrial-dependent while loss of mitochondrial membrane potential was not found in Met stem-treated cells. In addition, the expression levels of AIFM1, CASP9, CFLAR, and IGF1R were altered after treatment. Decreased BCL-2 expression was found in treated cells while BAX and caspases' expression was upregulated or maintained. All extracts caused perturbation of cell cycle at S phase by dysregulating the expression of cell cycle regulators such as CDKs and cyclins. Our findings indicate that P. nodiflora inhibits MCF-7 cells by inducing apoptosis and perturbing cell cycle.

Inhibition and Substrate Specificity Properties of FKBP22 from a Psychrotrophic Bacterium, Shewanella sp. SIB1.

SIB1 FKBP22 is a peptidyl prolyl cis-trans isomerase (PPIase) member from a psychrotrophic bacterium, Shewanella sp. SIB1, consisting of N- and C-domains responsible for dimerization and catalytic PPIase activity, respectively. This protein was assumed to be involved in cold adaptation of SIB1 cells through its dual activity of PPIase activity and chaperone like-function. Nevertheless, the catalytic inhibition by FK506 and its substrate specificity remain unknown. Besides, ability of SIB1 FKBP22 to inhibit phosphatase activity of calcinuerin is also interesting to be studied since it may reflect wider cellular functions of SIB1 FKBP22. In this study, we found that wild type (WT) SIB1 FKBP22 bound to FK506 with IC50 of 77.55 nM. This value is comparable to that of monomeric mutants (NNC-FKBP22, C-domain+ and V37R/L41R mutants), yet significantly higher than that of active site mutant (R142A). In addition, WT SIB1 FKBP22 and monomeric variants were found to prefer hydrophobic residues preceding proline. Meanwhile, R142A mutant has wider preferences on bulkier hydrophobic residues due to increasing hydrophobicity and binding pocket space. Surprisingly, in the absence of FK506, SIB1 FKBP22 and its variants inhibited, with the exception of N-domain, calcineurin phosphatase activity, albeit low. The inhibition of SIB1 FKBP22 by FK506 is dramatically increased in the presence of FK506. Altogether, we proposed that local structure at substrate binding pocket of C-domain plays crucial role for the binding of FK506 and peptide substrate preferences. In addition, C-domain is essential for inhibition, while dimerization state is important for optimum inhibition through efficient binding to calcineurin.

Chemical composition and cytotoxic properties of Clinacanthus nutans root extracts.

CONTEXT: Clinacanthus nutans Lindau (Acanthaceae) is a medicinal plant that has been reported to have anti-inflammatory, antiviral, antimicrobial and antivenom activities. In Malaysia, it has been widely claimed to be effective in various cancer treatments but scientific evidence is lacking. OBJECTIVE: This study investigates the chemical constituents, anti-proliferative, and apoptotic properties of C. nutans root extracts. MATERIALS AND METHODS: The roots were subjected to solvent extraction using methanol and ethyl acetate. The anti-proliferative effects of root extracts were tested at the concentrations of 10 to 50 µg/mL on MCF-7 and HeLa by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay for 72 h. Morphological changes were observed under light microscope. Pro-apoptotic effects of root extracts were examined using flow cytometric analysis and RT-PCR. The chemical compositions of root extracts were detected using GC-MS. RESULTS: The proliferation of MCF-7 cells was inhibited with the IC50 values of 35 and 30 µg/mL, respectively, for methanol and ethyl acetate root extracts. The average inhibition of HeLa cells was ∼25%. Induction of apoptosis in MCF-7 was supported by chromatin condensation, down-regulation of BCL2 and unaltered expression of BAX. However, only ethyl acetate extract caused the loss of mitochondrial membrane potential. GC-MS analysis revealed the roots extracts were rich with terpenoids and phytosterols. DISCUSSION AND CONCLUSIONS: The results demonstrated that root extracts promote apoptosis by suppressing BCL2 via mitochondria-dependent or independent manner. The identified compounds might work solely or cooperatively in regulating apoptosis. However, further studies are required to address this.

Separation and identification of volatile compounds from liquid cultures of Trichoderma harzianum by GC-MS using three different capillary columns.

A simple, fast, repeatable and less laborious sample preparation protocol was developed and applied for the analysis of biocontrol fungus Trichoderma harzianum strain FA1132 by using gas chromatography-mass spectrometry. The match factors for sample spectra with respect to the mass spectra library of fungal volatile compounds were determined and used to study the complex hydrocarbons and other volatile compounds, which were separated by using different capillary columns with nonpolar, medium polar and high polar stationary phases. To date, more than 278 volatile compounds (with spectral match factor at least 90%) such as normal saturated hydrocarbons (C7-C30), cyclohexane, cyclopentane, fatty acids, alcohols, esters, sulfur-containing compounds, simple pyrane and benzene derivatives have been identified. Most of these compounds have not previously been reported. The method described in this paper is a more convenient research tool for the detection of volatile compounds from the cultures of T. harzianum.

Molecular cloning of an O-methyltransferase from adventitious roots of Carapichea ipecacuanha.

Carapichea ipecacuanha produces various emetine-type alkaloids, known as ipecac alkaloids, which have long been used as expectorants, emetics, and amebicides. In this study, we isolated an O-methyltransferase cDNA from this medicinal plant. The encoded protein (CiOMT1) showed 98% sequence identity to IpeOMT2, which catalyzes the 7'-O-methylation of 7'-O-demethylcephaeline to form cephaeline at the penultimate step of emetine biosynthesis (Nomura and Kutchan, J. Biol. Chem., 285, 7722-7738 (2010)). Recombinant CiOMT1 showed both 7'-O-methylation and 6'-O-methylation activities at the last two steps of emetine biosynthesis. This indicates that small differences in amino acid residues are responsible for distinct regional methylation specificities between IpeOMT2 and CiOMT1, and that CiOMT1 might contribute to two sequential O-methylation steps from 7'-O-demethylcephaeline to emetine.