Improved 13C metabolic flux analysis in Escherichia coli metabolism: application of a high-resolution MS (GC-EI-QTOF) for comprehensive assessment of MS/MS fragments.

Gas chromatography-tandem mass spectrometry with electron ionization (GC-EI-MS/MS) provides rich information on stable-isotope labeling for 13C-metabolic flux analysis (13C-MFA). To pave the way for the routine application of tandem MS data for metabolic flux quantification, we aimed to compile a comprehensive library of GC-EI-MS/MS fragments of tert-butyldimethylsilyl (TBDMS) derivatized proteinogenic amino acids. First, we established an analytical workflow that combines high-resolution gas chromatography-quadrupole time-of-flight mass spectrometry and fully 13C-labeled biomass to identify and structurally elucidate tandem MS amino acid fragments. Application of the high-mass accuracy MS procedure resulted into the identification of 129 validated precursor-product ion pairs of 13 amino acids with 30 fragments being accepted for 13C-MFA. The practical benefit of the novel tandem MS data was demonstrated by a proof-of-concept study, which confirmed the importance of the compiled library for high-resolution 13C-MFA. ONE SENTENCE SUMMARY: An analytical workflow that combines high-resolution mass spectrometry (MS) and fully 13C-labeled biomass to identify and structurally elucidate tandem MS amino acid fragments, which provide positional information and therefore offering significant advantages over traditional MS to improve 13C-metabolic flux analysis.

Cell type matters: competence for alkaloid metabolism differs in two seed-derived cell strains of Catharanthus roseus.

Since the discovery of the anticancer drugs vinblastine and vincristine, Catharanthus roseus has been intensively studied for biosynthesis of several terpene indole alkaloids (TIAs). Due to their low abundance in plant tissues at a simultaneously high demand, modes of production alternative to conventional extraction are mandatory. Plant cell fermentation might become one of these alternatives, yet decades of research have shown limited success to certain product classes, leading to the question: how to preserve the intrinsic ability to produce TIAs (metabolic competence) in cell culture? We used the strategy to use the developmental potency of mature embryos to generate such strains. Two cell strains (C1and C4) from seed embryos of Catharanthus roseus were found to differ not only morphologically, but also in their metabolic competence. This differential competence became manifest not only under phytohormone elicitation, but also upon feeding with alkaloid pathway precursors. The more active strain C4 formed larger cell aggregates and was endowed with longer mitochondria. These cellular features were accompanied by higher alkaloid accumulation in response to methyl jasmonate (MeJA) elicitation. The levels of catharanthine could be increased significantly, while the concurrent vindoline branch of the pathway was blocked, such that no bisindole alkaloids were detectable. By feeding vindoline to MeJA-elicited C4 cells, vincristine became detectable; however, only to marginal amounts. In conclusion, these results show that cultured cells are not "de-differentiated", but can differ in metabolic competence. In addition to elicitation and precursor feeding, the cellular properties of the "biomatter" are highly relevant for the success of plant cell fermentation.

Gain time to adapt: How sorghum acquires tolerance to salinity.

Salinity is a global environmental threat to agricultural production and food security around the world. To delineate salt-induced damage from adaption events we analysed a pair of sorghum genotypes which are contrasting in their response to salt stress with respect to physiological, cellular, metabolomic, and transcriptional responses. We find that the salt-tolerant genotype Della can delay the transfer of sodium from the root to the shoot, more swiftly deploy accumulation of proline and antioxidants in the leaves and transfer more sucrose to the root as compared to its susceptible counterpart Razinieh. Instead Razinieh shows metabolic indicators for a higher extent photorespiration under salt stress. Following sodium accumulation by a fluorescent dye in the different regions of the root, we find that Della can sequester sodium in the vacuoles of the distal elongation zone. The timing of the adaptive responses in Della leaves indicates a rapid systemic signal from the roots that is travelling faster than sodium itself. We arrive at a model where resistance and susceptibility are mainly a matter of temporal patterns in signalling.

Switching cell fate by the actin-auxin oscillator in Taxus: cellular aspects of plant cell fermentation.

KEY MESSAGE: Paclitaxel synthesis in Taxus cells correlates with a cell-fate switch that leads to vacuoles of a glossy appearance and vermiform mitochondria. This switch depends on actin and apoplastic respiratory burst. Plant cell fermentation, the production of valuable products in plant cell culture, has great potential as sustainable alternative to the exploitation of natural resources for compounds of pharmaceutical interest. However, the success of this approach has remained limited, because the cellular aspects of metabolic competence are mostly unknown. The production of the anti-cancer alkaloid Paclitaxel has been, so far, the most successful case for this approach. In the current work, we map cellular aspects of alkaloid synthesis in cells of Taxus chinensis using a combination of live-cell imaging, quantitative physiology, and metabolite analysis. We show evidence that metabolic potency correlates with a differentiation event giving rise to cells with large vacuoles with a tonoplast that is of a glossy appearance, agglomerations of lipophilic compounds, and multivesicular bodies that fuse with the plasma membrane. Cellular features of these glossy cells are bundled actin, more numerous peroxisomes, and vermiform mitochondria. The incidence of glossy cells can be increased by aluminium ions, and this increase is significantly reduced by the actin inhibitor Latrunculin B, and by diphenylene iodonium, a specific inhibitor of the NADPH oxidase Respiratory burst oxidase Homologue (RboH). It is also reduced by the artificial auxin Picloram. This cellular fingerprint matches the implications of a model, where the differentiation into the glossy cell type is regulated by the actin-auxin oscillator that in plant cells acts as dynamic switch between growth and defence.

Genome-wide association study on metabolite accumulation in a wild barley NAM population reveals natural variation in sugar metabolism.

Metabolites play a key role in plants as they are routing plant developmental processes and are involved in biotic and abiotic stress responses. Their analysis can offer important information on the underlying processes. Regarding plant breeding, metabolite concentrations can be used as biomarkers instead of or in addition to genetic markers to predict important phenotypic traits (metabolic prediction). In this study, we applied a genome-wide association study (GWAS) in a wild barley nested association mapping (NAM) population to identify metabolic quantitative trait loci (mQTL). A set of approximately 130 metabolites, measured at early and late sampling dates, was analysed. For four metabolites from the early and six metabolites from the late sampling date significant mQTL (grouped as 19 mQTL for the early and 25 mQTL for the late sampling date) were found. Interestingly, all of those metabolites could be classified as sugars. Sugars are known to be involved in signalling, plant growth and plant development. Sugar-related genes, encoding mainly sugar transporters, have been identified as candidate genes for most of the mQTL. Moreover, several of them co-localized with known flowering time genes like Ppd-H1, HvELF3, Vrn-H1, Vrn-H2 and Vrn-H3, hinting on the known role of sugars in flowering. Furthermore, numerous disease resistance-related genes were detected, pointing to the signalling function of sugars in plant resistance. An mQTL on chromosome 1H in the region of 13 Mbp to 20 Mbp stood out, that alone explained up to 65% of the phenotypic variation of a single metabolite. Analysis of family-specific effects within the diverse NAM population showed the available natural genetic variation regarding sugar metabolites due to different wild alleles. The study represents a step towards a better understanding of the genetic components of metabolite accumulation, especially sugars, thereby linking them to biological functions in barley.

Can metabolic prediction be an alternative to genomic prediction in barley?

Like other crop species, barley, the fourth most important crop worldwide, suffers from the genetic bottleneck effect, where further improvements in performance through classical breeding methods become difficult. Therefore, indirect selection methods are of great interest. Here, genomic prediction (GP) based on 33,005 SNP markers and, alternatively, metabolic prediction (MP) based on 128 metabolites with sampling at two different time points in one year, were applied to predict multi-year agronomic traits in the nested association mapping (NAM) population HEB-25. We found prediction abilities of up to 0.93 for plant height with SNP markers and of up to 0.61 for flowering time with metabolites. Interestingly, prediction abilities in GP increased after reducing the number of incorporated SNP markers. The estimated effects of GP and MP were highly concordant, indicating MP as an interesting alternative to GP, being able to reflect a stable genotype-specific metabolite profile. In MP, sampling at an early developmental stage outperformed sampling at a later stage. The results confirm the value of GP for future breeding. With MP, an interesting alternative was also applied successfully. However, based on our results, usage of MP alone cannot be recommended in barley. Nevertheless, MP can assist in unravelling physiological pathways for the expression of agronomically important traits.

Shoot tip culture: a step towards 13C metabolite flux analysis of sink leaf metabolism.

BACKGROUND: Better understanding of the physiological and metabolic status of plants can only be obtained when metabolic fluxes are accurately assessed in a growing plant. Steady state 13C-MFA has been established as a routine method for analysis of fluxes in plant primary metabolism. However, the experimental system needs to be improved for continuous carbon enrichment from labelled sugars into metabolites for longer periods until complex secondary metabolism reaches steady state. RESULTS: We developed an in vitro plant culture strategy by using peppermint as a model plant with minimizing unlabelled carbon fixation where growing shoot tip was strongly dependent on labelled glucose for their carbon necessity. We optimized the light condition and detected the satisfactory phenotypical growth under the lower light intensity. Total volatile terpenes were also highest at the same light. Analysis of label incorporation into pulegone monoterpene after continuous U-13C6 glucose feeding revealed nearly 100% 13C, even at 15 days after first leaf visibility (DALV). Label enrichment was gradually scrambled with increasing light intensity and leaf age. This study was validated by showing similar levels of label enrichment in proteinogenic amino acids. The efficiency of this method was also verified in oregano. CONCLUSIONS: Our shoot tip culture depicted a method in achieving long term, stable and a high percentage of label accumulation in secondary metabolites within a fully functional growing plant system. It recommends the potential application for the investigations of various facets of plant metabolism by steady state 13C-MFA. The system also provides a greater potential to study sink leaf metabolism. Overall, we propose a system to accurately describe complex metabolic phenotypes in a growing plant.

Corrigendum: Physiological and Proteomic Analysis of the Rice Mutant cpm2 Suggests a Negative Regulatory Role of Jasmonic Acid in Drought Tolerance.

[This corrects the article on p. 1903 in vol. 8, PMID: 29250082.].

Physiological and Proteomic Analysis of the Rice Mutant cpm2 Suggests a Negative Regulatory Role of Jasmonic Acid in Drought Tolerance.

It is widely known that numerous adaptive responses of drought-stressed plants are stimulated by chemical messengers known as phytohormones. Jasmonic acid (JA) is one such phytohormone. But there are very few reports revealing its direct implication in drought related responses or its cross-talk with other phytohormones. In this study, we compared the morpho-physiological traits and the root proteome of a wild type (WT) rice plant with its JA biosynthesis mutant coleoptile photomorphogenesis 2 (cpm2), disrupted in the allene oxide cyclase (AOC) gene, for insights into the role of JA under drought. The mutant had higher stomatal conductance, higher water use efficiency and higher shoot ABA levels under severe drought as compared to the WT. Notably, roots of cpm2 were better developed compared to the WT under both, control and drought stress conditions. Root proteome was analyzed using the Tandem Mass Tag strategy to better understand this difference at the molecular level. Expectedly, AOC was unique but notably highly abundant under drought in the WT. Identification of other differentially abundant proteins (DAPs) suggested increased energy metabolism (i.e., increased mobilization of resources) and reactive oxygen species scavenging in cpm2 under drought. Additionally, various proteins involved in secondary metabolism, cell growth and cell wall synthesis were also more abundant in cpm2 roots. Proteome-guided transcript, metabolite, and histological analyses provided further insights into the favorable adaptations and responses, most likely orchestrated by the lack of JA, in the cpm2 roots. Our results in cpm2 are discussed in the light of JA crosstalk to other phytohormones. These results together pave the path for understanding the precise role of JA during drought stress in rice.

Reference genes for accurate gene expression analyses across different tissues, developmental stages and genotypes in rice for drought tolerance.

BACKGROUND: Quantitative reverse transcription PCR (qRT-PCR) has been routinely used to quantify gene expression level. This technique determines the expression of a target gene by comparison to an internal control gene uniformly expressed among the samples analyzed. The reproducibility and reliability of the results depend heavily on the reference genes used. To achieve successful gene expression analyses for drought tolerance studies in rice, reference gene selection should be based on consistency in expression across variables. We aimed to provide reference genes that would be consistent across different tissues, developmental stages and genotypes of rice and hence improve the quality of data in qRT-PCR analysis. FINDINGS: Ten candidate reference genes were screened from four ubiquitously expressed gene families by analyzing public microarray data sets that included profiles of multiple organs, developmental stages, and water availability status in rice. These genes were evaluated through qRT-PCR experiments with a rigorous statistical analysis to determine the best reference genes. A ubiquitin isogene showed the best gene expression stability as a single reference gene, while a 3-gene combination of another ubiquitin and two cyclophilin isogenes was the best reference gene combination. Comparison between the qRT-PCR and in-house microarray data on roots demonstrated reliability of the identified reference genes to monitor the differential expression of drought-related candidate genes. CONCLUSIONS: Specific isogenes from among the regularly used gene families were identified for use in qRT-PCR-based analyses for gene expression in studies on drought tolerance in rice. These were stable across variables of treatment, genotype, tissue and growth stage. A single gene and/or a three gene set analysis is recommended, based on the resources available.

Total Soluble Protein Extraction for Improved Proteomic Analysis of Transgenic Rice Plant Roots.

With the advent of high-throughput platforms, proteomics has become a powerful tool to search for plant gene products of agronomic relevance. Protein extractions using multistep protocols have been shown to be effective to achieve better proteome profiles than simple, single-step extractions. These protocols are generally efficient for above ground tissues such as leaves. However, each step leads to loss of some amount of proteins. Additionally, compounds such as proteases in the plant tissues lead to protein degradation. While protease inhibitor cocktails are available, these alone do not seem to suffice when roots are included in the plant sample. This is obvious given the lack of high molecular weight (HMW) proteins obtained from samples that include root tissue. For protein/proteome analysis of transgenic plant roots or of seedlings, which include root tissue, such pronounced protein degradation is especially undesirable. A facile protein extraction protocol is presented, which ensures that despite the inclusion of root tissues there is minimal loss in total protein components.

Action of multiple intra-QTL genes concerted around a co-localized transcription factor underpins a large effect QTL.

Sub-QTLs and multiple intra-QTL genes are hypothesized to underpin large-effect QTLs. Known QTLs over gene families, biosynthetic pathways or certain traits represent functional gene-clusters of genes of the same gene ontology (GO). Gene-clusters containing genes of different GO have not been elaborated, except in silico as coexpressed genes within QTLs. Here we demonstrate the requirement of multiple intra-QTL genes for the full impact of QTL qDTY12.1 on rice yield under drought. Multiple evidences are presented for the need of the transcription factor 'no apical meristem' (OsNAM12.1) and its co-localized target genes of separate GO categories for qDTY12.1 function, raising a regulon-like model of genetic architecture. The molecular underpinnings of qDTY12.1 support its effectiveness in further improving a drought tolerant genotype and for its validity in multiple genotypes/ecosystems/environments. Resolving the combinatorial value of OsNAM12.1 with individual intra-QTL genes notwithstanding, identification and analyses of qDTY12.1has fast-tracked rice improvement towards food security.

Variation in primary metabolites in parental and near-isogenic lines of the QTL qDTY12.1 : altered roots and flag leaves but similar spikelets of rice under drought.

There is a widespread consensus that drought will mostly affect present and future agriculture negatively. Generating drought-tolerant crops is thus a high priority. However complicated the underlying genetic and regulatory networks for differences in plant performance under stress are, they would be reflected in straightforward differences in primary metabolites. This is because primary metabolites such as amino acids and sugars form the building blocks of all pathways and processes for growth, development, reproduction, and environmental responses. Comparison of such differences was undertaken between the parental line and a near-isogenic line of qDTY12.1 , a QTL for rice yield under drought. The comparison was informative regarding the effect of the QTL in three genetic backgrounds: donor, recipient, and improved recipient, thus illustrating the gene × gene (G × G) interactions. Such a comparison when extended to well-watered and drought conditions illustrated the gene × environment (G × E) interactions. Assessment of such G × G and G × E responses in roots, flag leaves, and spikelets added a yet more informative dimension of tissue-specific responses to drought, mediated by qDTY12.1 . Data on variation in primary metabolites subjected to ANOVA, Tukey's test, Welch's t test, and PCA underscored the importance of the roots and demonstrated concordance between variation in metabolites and morpho-physiological responses to drought. Results suggested that for gainful insights into rice yield under drought, rather than vegetative stage drought tolerance, multiple tissues and genotypes must be assessed at the reproductive stage to avoid misleading conclusions about using particular metabolites or related genes and proteins as candidates or markers for drought tolerance.

Protein SUMOylation and plant abiotic stress signaling: in silico case study of rice RLKs, heat-shock and Ca(2+)-binding proteins.

Plants respond to stress conditions through early stress-response factors (ESRF), which serve the function of stress sensing and/or signal transduction. These mainly comprise qualitative and/or quantitative flux in the redox molecules, calcium ions (Ca(2+)), phosphatidic acid, hexose sugars and phytohormones. The role of resident proteins such as phytohormone receptors and G-proteins as first messengers under stress is well established. Yet, within the modern omics context, most of the stress response at the protein level is injudiciously attributed to substantial up- or down-regulation of expression measured at the RNA or protein level. Proteins such as kinases and transcription factors (TFs) that exhibit cascade effects are primary candidates for studies in plant stress tolerance. However, resident-protein post-translational modification (PTM), specifically in response to particular conditions such as stress, is a candidate for immediate and potent 'quick reaction force' (QRF) kind of effects. Stress-mediated SUMOylation of TFs and other proteins have been observed. SUMOylation can change the rate of activity, function or location of the modified protein. Early SUMOylation of resident proteins can act in the stress signal transduction or in adaptive response. Here, we consider brief background information on ESRFs to establish the crosstalk between these factors that impinge on PTMs. We then illustrate connections of protein SUMOylation to phytohormones and TFs. Finally, we present results of an in silico analysis of rice Receptor-Like Kinases, heat-shock and calcium-binding proteins to identify members of these gene families, whose basal expression under drought but potential SUMOylation presents them as QRF candidates for roles in stress signaling/response.