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.

IntroductionFrost 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.ObjectivesThis 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.MethodsWe 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.ResultsThe 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.ConclusionOur study suggests that metabolomics and lipidomics markers could be used as an alternative phenotyping method to discriminate wheat varieties with differences in cold acclimation.Electronic supplementary materialThe online version of this article (10.1007/s11306-019-1606-2) contains supplementary material, which is available to authorized users.

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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.

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.

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.

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29 30 WORD COUNT 31 18,932 32 2 TITLE 33 Arabidopsis REIL proteins activate ribosome biogenesis during cold acclimation 34 Running Title 35 REIL proteins activate ribosome biogenesis 36 Highlight of this study (summarizing sentence) (27 words) 37 REIL proteins affect paralog composition of eukaryotic ribosomes and suppress accumulation of 38 43S-preinitiation and pre-60S-maturation complexes, suggesting functions of ribosome 39 heterogeneity and biogenesis in plant cold acclimation. 40 Abstract (150 words) 41 Arabidopsis REIL proteins are cytosolic ribosomal 60S-biogenesis factors. After shift to 10°C, reil 42 mutants deplete and slowly replenish non-translating eukaryotic ribosome complexes of root 43 tissue, while tightly controlling the balance of non-translating 40S-and 60S-subunits. Reil 44 mutations compensate by hyper-accumulation of non-translating subunits at steady-state 45 temperature; after cold-shift, a KCl-sensitive 80S sub-fraction remains depleted. We infer that 46 Arabidopsis buffers fluctuating translation by pre-existing non-translating ribosomes before de 47 novo synthesis meets temperature-induced demands. Reil1 reil2 double mutants accumulate 43S-48 preinitiation and pre-60S-maturation complexes and have altered paralog composition of 49 ribosomal proteins in non-translating complexes. With few exceptions, e.g. RPL3B and RPL24C, 50 these changes are not under transcriptional control. Our study suggests requirement of de novo 51 synthesis of eukaryotic ribosomes for long-term cold acclimation, feedback control of NUC2 and 52 eIF3C2 transcription and links new proteins, AT1G03250, AT5G60530, to plant ribosome 53 biogenesis. We propose that Arabidopsis requires biosynthesis of specialized ribosomes for cold 54 acclimation. 55 Key words (6-10, in alphabetical order) 56 abiotic stress, Arabidopsis, cold acclimation, proteomics, REI-LIKE proteins, ribosome 57 biogenesis, roots, system analysis, transcriptomics 58 59 60 61The Arabidopsis thaliana Col-0 (Arabidopsis) REI1-LIKE (REIL) proteins, REIL1 62 (At4g31420) and REIL2 (At2g24500) are homologs of the yeast Rei1 (YBR267W) and the paralog 63 Reh1 (YLR387C) proteins. In yeast, both the Rei1 and Reh1 proteins function as ribosome 64 biogenesis factors that participate either in parallel or sequentially in the late ribosome biogenesis 65 step of cytoplasmic 60S ribosomal subunit maturation (Greber et al., 2012; Greber et al., 2016). 66 Aside from this function, these proteins are required to maintain growth at suboptimal or cold 67 temperatures. The ∆rei1 mutant is cold sensitive already at moderately suboptimal temperatures 68 of yeast. The ∆rei1 ∆reh1 double mutant is even more cold sensitive, while the yeast ∆reh1 69 mutation alone has no effect on growth in the cold (Iwase and Toh-e, 2004; Lebreton et al., 2006; 70 Parnell and Bass, 2009). Heterologous expression of Arabidopsis REIL1, but not of REIL2 partly 71 complements the cold sensitivity of the yeast ∆rei1 mutant (Schmidt et al., 2013). The Arabidopsis 72 REIL paralogs differ in st...