NMR spectroscopy analysis reveals differential metabolic responses in arabidopsis roots and leaves treated with a cytokinesis inhibitor.

In plant cytokinesis, de novo formation of a cell plate evolving into the new cell wall partitions the cytoplasm of the dividing cell. In our earlier chemical genomics studies, we identified and characterized the small molecule endosidin-7, that specifically inhibits callose deposition at the cell plate, arresting late-stage cytokinesis in arabidopsis. Endosidin-7 has emerged as a very valuable tool for dissecting this essential plant process. To gain insights regarding its mode of action and the effects of cytokinesis inhibition on the overall plant response, we investigated the effect of endosidin-7 through a nuclear magnetic resonance spectroscopy (NMR) metabolomics approach. In this case study, metabolomics profiles of arabidopsis leaf and root tissues were analyzed at different growth stages and endosidin-7 exposure levels. The results show leaf and root-specific metabolic profile changes and the effects of endosidin-7 treatment on these metabolomes. Statistical analyses indicated that the effect of endosidin-7 treatment was more significant than the developmental impact. The endosidin-7 induced metabolic profiles suggest compensations for cytokinesis inhibition in central metabolism pathways. This study further shows that long-term treatment of endosidin-7 profoundly changes, likely via alteration of hormonal regulation, the primary metabolism of arabidopsis seedlings. Hormonal pathway-changes are likely reflecting the plant's responses, compensating for the arrested cell division, which in turn are leading to global metabolite modulation. The presented NMR spectral data are made available through the Metabolomics Workbench, providing a reference resource for the scientific community.

PDC1, a pyruvate/α-ketoacid decarboxylase, is involved in acetaldehyde, propanal and pentanal biosynthesis in melon (Cucumis melo L.) fruit.

Plant pyruvate decarboxylases (PDC) catalyze the decarboxylation of pyruvate to form acetaldehyde and CO2 and are well known to play a key role in energy supply via fermentative metabolism in oxygen-limiting conditions. In addition to their role in fermentation, plant PDCs have also been hypothesized to be involved in aroma formation although, to date, there is no direct biochemical evidence for this function. We investigated the role of PDCs in fruit volatile biosynthesis, and identified a melon pyruvate decarboxylase, PDC1, that is highly expressed in ripe fruits. In vitro biochemical characterization of the recombinant PDC1 enzyme showed that it could not only decarboxylate pyruvate, but that it also had significant activity toward other straight- and branched-chain α-ketoacids, greatly expanding the range of substrates previously known to be accepted by the plant enzyme. RNAi-mediated transient and stable silencing of PDC1 expression in melon showed that this gene is involved in acetaldehyde, propanal and pentanal production, while it does not contribute to branched-chain amino acid (BCAA)-derived aldehyde biosynthesis in melon fruit. Importantly, our results not only demonstrate additional functions for the PDC enzyme, but also challenge the long standing hypothesis that PDC is involved in BCAA-derived aldehyde formation in fruit.

'Bartlett' pear fruit (Pyrus communis L.) ripening regulation by low temperatures involves genes associated with jasmonic acid, cold response, and transcription factors.

Low temperature (LT) treatments enhance ethylene production and ripening rate in the European pear (Pyrus communis L.). However, the underlying molecular mechanisms are not well understood. This study aims to identify genes responsible for ripening enhancement by LT. To this end, the transcriptome of 'Bartlett' pears treated with LT (0°C or 10°C for up to 14 d), which results in faster ripening, and control pears without conditioning treatment was analyzed. LT conditioned pears reached eating firmness (18N) in 6 d while control pears took about 12 d when left to ripen at 20°C. We identified 8,536 differentially expressed (DE) genes between the 0°C-treated and control fruit, and 7,938 DE genes between the 10°C-treated and control fruit. In an attempt to differentiate temperature-induced vs. ethylene-responsive pathways, we also monitored gene expression in fruit sequentially treated with 1-MCP then exposed to low temperature. This analysis revealed that genes associated with jasmonic acid biosynthesis and signaling, as well as the transcription factors TCP9a, TCP9b, CBF1, CBF4, AGL24, MYB1R1, and HsfB2b could be involved in the LT-mediated enhancement of ripening independently or upstream of ethylene.

HS-SPME-GC-MS/MS Method for the Rapid and Sensitive Quantitation of 2-Acetyl-1-pyrroline in Single Rice Kernels.

Demand for aromatic rice varieties (e.g., Basmati) is increasing in the US. Aromatic varieties typically have elevated levels of the aroma compound 2-acetyl-1-pyrroline (2AP). Due to its very low aroma threshold, analysis of 2AP provides a useful screening tool for rice breeders. Methods for 2AP analysis in rice should quantitate 2AP at or below sensory threshold level, avoid artifactual 2AP generation, and be able to analyze single rice kernels in cases where only small sample quantities are available (e.g., breeding trials). We combined headspace solid phase microextraction with gas chromatography tandem mass spectrometry (HS-SPME-GC-MS/MS) for analysis of 2AP, using an extraction temperature of 40 °C and a stable isotopologue as internal standard. 2AP calibrations were linear between the concentrations of 53 and 5380 pg/g, with detection limits below the sensory threshold of 2AP. Forty-eight aromatic and nonaromatic, milled rice samples from three harvest years were screened with the method for their 2AP content, and overall reproducibility, observed for all samples, ranged from 5% for experimental aromatic lines to 33% for nonaromatic lines.

A transcriptome approach towards understanding the development of ripening capacity in 'Bartlett' pears (Pyrus communis L.).

BACKGROUND: The capacity of European pear fruit (Pyrus communis L.) to ripen after harvest develops during the final stages of growth on the tree. The objective of this study was to characterize changes in 'Bartlett' pear fruit physico-chemical properties and transcription profiles during fruit maturation leading to attainment of ripening capacity. RESULTS: The softening response of pear fruit held for 14 days at 20 °C after harvest depended on their maturity. We identified four maturity stages: S1-failed to soften and S2- displayed partial softening (with or without ET-ethylene treatment); S3 - able to soften following ET; and S4 - able to soften without ET. Illumina sequencing and Trinity assembly generated 68,010 unigenes (mean length of 911 bp), of which 32.8 % were annotated to the RefSeq plant database. Higher numbers of differentially expressed transcripts were recorded in the S3-S4 and S1-S2 transitions (2805 and 2505 unigenes, respectively) than in the S2-S3 transition (2037 unigenes). High expression of genes putatively encoding pectin degradation enzymes in the S1-S2 transition suggests pectic oligomers may be involved as early signals triggering the transition to responsiveness to ethylene in pear fruit. Moreover, the co-expression of these genes with Exps (Expansins) suggests their collaboration in modifying cell wall polysaccharide networks that are required for fruit growth. K-means cluster analysis revealed that auxin signaling associated transcripts were enriched in cluster K6 that showed the highest gene expression at S3. AP2/EREBP (APETALA 2/ethylene response element binding protein) and bHLH (basic helix-loop-helix) transcripts were enriched in all three transition S1-S2, S2-S3, and S3-S4. Several members of Aux/IAA (Auxin/indole-3-acetic acid), ARF (Auxin response factors), and WRKY appeared to play an important role in orchestrating the S2-S3 transition. CONCLUSIONS: We identified maturity stages associated with the development of ripening capacity in 'Bartlett' pear, and described the transcription profile of fruit at these stages. Our findings suggest that auxin is essential in regulating the transition of pear fruit from being ethylene-unresponsive (S2) to ethylene-responsive (S3), resulting in fruit softening. The transcriptome will be helpful for future studies about specific developmental pathways regulating the transition to ripening.

Effect of ethylene and temperature conditioning on sensory attributes and chemical composition of 'Comice' pears.

'Comice' is among the pear varieties most difficult to ripen after harvest. Ethylene, cold temperature, and intermediate (10 °C) temperature conditioning have been successfully used to stimulate the ability of 'Comice' pears to ripen. However, the sensory quality of pears stimulated to ripen by different conditioning treatments has not been evaluated. In this study, a descriptive sensory analysis of 'Comice' pears conditioned to soften to 27, 18, and 9 N firmness with ethylene exposure for 3 or 1 days, storage at 0 °C for 25 or 15 days, or storage at 10 °C for 10 days was performed. Sensory attributes were then related to changes in chemical composition, including volatile components, water-soluble polyuronides, soluble solids content (SSC), and titratable acidity (TA). The sensory profile of fruit conditioned with ethylene was predominant in fibrous texture and low in fruity and pear aroma. Fruit conditioned at 0 °C was described as crunchy at 27 and 18 N firmness and became juicy at 9 N firmness. Fruit conditioned at 0 °C produced the highest quantity of alcohols and fewer esters than fruit conditioned at 10 °C, and they had higher fruity and pear aroma than fruit conditioned with ethylene, but lower than fruit conditioned at 10 °C. Fruit held at 10 °C were predominant in fruity and pear aroma and had the highest concentration of esters. Water-soluble polyuronides were strongly, positively correlated (r > 0.9) with sensory attributes generally associated with ripeness, including juiciness, butteriness, and sweetness and negatively correlated (r > -0.9) with sensory attributes generally associated with the unripe stage, such as firmness and crunchiness. However, water-soluble polyuronides were not significantly different among conditioning treatments. Sensory sweetness was not significantly correlated with SSC, but TA and SSC/TA were significantly correlated with sensory tartness. However, there were no significant differences among the conditioning treatments in sweet or sour taste perception when the fruit fully softened. The results indicate that the various methods of conditioning 'Comice' pear fruits for ripening had different effects on their sensory and chemical properties that may influence their sensory quality.

An integrated approach for flavour quality evaluation in muskmelon (Cucumis melo L. reticulatus group) during ripening.

Numerous and diverse physiological changes occur during fruit ripening and maturity at harvest is one of the key factors influencing the flavour quality of fruits. The effect of ripening on chemical composition, physical parameters and sensory perception of three muskmelon (Cucumis melo L. reticulatus group) cultivars was evaluated. Significant correlations emerging from this extensive data set are discussed in the context of identifying potential targets for melon sensory quality improvement. A portable ultra-fast gas-chromatograph coupled with a surface acoustic wave sensor (UFGC-SAW) was also used to monitor aroma volatile concentrations during fruit ripening and evaluated for its ability to predict the sensory perception of melon flavour. UFGC-SAW analysis allowed the discrimination of melon maturity stage based on six measured peaks, whose abundance was positively correlated to maturity-specific sensory attributes. Our findings suggest that this technology shows promise for future applications in rapid flavour quality evaluation.

Stomatal density and metabolic determinants mediate salt stress adaptation and water use efficiency in basil (Ocimum basilicum L.).

Increasing salinity tolerance and water-use efficiency in crop plants are two major challenges that agriculture must face in the next decades. Many physiological mechanisms and molecular components mediating crop response to environmental stresses have been identified. However, the functional inter-links between stress adaptation responses have not been completely understood. Using two basil cultivars (Napoletano and Genovese) with contrasting ability to respond to salt stress, here we demonstrate that reduced stomatal density, high ascorbate level and polyphenol oxidase (PPO) activity coordinately contribute to improve basil adaptation and water use efficiency (WUE) in saline environment. The constitutively reduced stomatal density was associated with a "delayed" accumulation of stress molecules (and growth inhibiting signals) such as abscisic acid (ABA) and proline, in the more tolerant Genovese. Leaf volatile profiling also revealed cultivar-specific patterns, which may suggest a role for the volatile phenylpropanoid eugenol and monoterpenes in conferring stress tolerance via antioxidant and signalling functions.

Fruit volatile analysis using an electronic nose.

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit aroma. Maturity at harvest is one of the key factors influencing the flavor quality of fruits and vegetables. The validation of robust methods that rapidly assess fruit maturity and aroma quality would allow improved management of advanced breeding programs, production practices and postharvest handling. Over the last three decades, much research has been conducted to develop so-called electronic noses, which are devices able to rapidly detect odors and flavors. Currently there are several commercially available electronic noses able to perform volatile analysis, based on different technologies. The electronic nose used in our work (zNose, EST, Newbury Park, CA, USA), consists of ultra-fast gas chromatography coupled with a surface acoustic wave sensor (UFGC-SAW). This technology has already been tested for its ability to monitor quality of various commodities, including detection of deterioration in apple; ripeness and rot evaluation in mango; aroma profiling of thymus species; C(6) volatile compounds in grape berries; characterization of vegetable oil and detection of adulterants in virgin coconut oil. This system can perform the three major steps of aroma analysis: headspace sampling, separation of volatile compounds, and detection. In about one minute, the output, a chromatogram, is produced and, after a purging cycle, the instrument is ready for further analysis. The results obtained with the zNose can be compared to those of other gas-chromatographic systems by calculation of Kovats Indices (KI). Once the instrument has been tuned with an alkane standard solution, the retention times are automatically converted into KIs. However, slight changes in temperature and flow rate are expected to occur over time, causing retention times to drift. Also, depending on the polarity of the column stationary phase, the reproducibility of KI calculations can vary by several index units. A series of programs and graphical interfaces were therefore developed to compare calculated KIs among samples in a semi-automated fashion. These programs reduce the time required for chromatogram analysis of large data sets and minimize the potential for misinterpretation of the data when chromatograms are not perfectly aligned. We present a method for rapid volatile compound analysis in fruit. Sample preparation, data acquisition and handling procedures are also discussed.

Label-free shotgun proteomics and metabolite analysis reveal a significant metabolic shift during citrus fruit development.

Label-free LC-MS/MS-based shot-gun proteomics was used to quantify the differential protein synthesis and metabolite profiling in order to assess metabolic changes during the development of citrus fruits. Our results suggested the occurrence of a metabolic change during citrus fruit maturation, where the organic acid and amino acid accumulation seen during the early stages of development shifted into sugar synthesis during the later stage of citrus fruit development. The expression of invertases remained unchanged, while an invertase inhibitor was up-regulated towards maturation. The increased expression of sucrose-phosphate synthase and sucrose-6-phosphate phosphatase and the rapid sugar accumulation suggest that sucrose is also being synthesized in citrus juice sac cells during the later stage of fruit development.

Role of aromatic aldehyde synthase in wounding/herbivory response and flower scent production in different Arabidopsis ecotypes.

Aromatic L-amino acid decarboxylases (AADCs) are key enzymes operating at the interface between primary and secondary metabolism. The Arabidopsis thaliana genome contains two genes, At2g20340 and At4g28680, encoding pyridoxal 5'-phosphate-dependent AADCs with high homology to the recently identified Petunia hybrida phenylacetaldehyde synthase involved in floral scent production. The At4g28680 gene product was recently biochemically characterized as an L-tyrosine decarboxylase (AtTYDC), whereas the function of the other gene product remains unknown. The biochemical and functional characterization of the At2g20340 gene product revealed that it is an aromatic aldehyde synthase (AtAAS), which catalyzes the conversion of phenylalanine and 3,4-dihydroxy-L-phenylalanine to phenylacetaldehyde and dopaldehyde, respectively. AtAAS knock-down and transgenic AtAAS RNA interference (RNAi) lines show significant reduction in phenylacetaldehyde levels and an increase in phenylalanine, indicating that AtAAS is responsible for phenylacetaldehyde formation in planta. In A. thaliana ecotype Columbia (Col-0), AtAAS expression was highest in leaves, and was induced by methyl jasmonate treatment and wounding. Pieris rapae larvae feeding on Col-0 leaves resulted in increased phenylacetaldehyde emission, suggesting that the emitted aldehyde has a defensive activity against attacking herbivores. In the ecotypes Sei-0 and Di-G, which emit phenylacetaldehyde as a predominant flower volatile, the highest expression of AtAAS was found in flowers and RNAi AtAAS silencing led to a reduction of phenylacetaldehyde formation in this organ. In contrast to ecotype Col-0, no phenylacetaldehyde accumulation was observed in Sei-0 upon wounding, suggesting that AtAAS and subsequently phenylacetaldehyde contribute to pollinator attraction in this ecotype.

Distinct roles of jasmonates and aldehydes in plant-defense responses.

BACKGROUND: Many inducible plant-defense responses are activated by jasmonates (JAs), C(6)-aldehydes, and their corresponding derivatives, produced by the two main competing branches of the oxylipin pathway, the allene oxide synthase (AOS) and hydroperoxide lyase (HPL) branches, respectively. In addition to competition for substrates, these branch-pathway-derived metabolites have substantial overlap in regulation of gene expression. Past experiments to define the role of C(6)-aldehydes in plant defense responses were biased towards the exogenous application of the synthetic metabolites or the use of genetic manipulation of HPL expression levels in plant genotypes with intact ability to produce the competing AOS-derived metabolites. To uncouple the roles of the C(6)-aldehydes and jasmonates in mediating direct and indirect plant-defense responses, we generated Arabidopsis genotypes lacking either one or both of these metabolites. These genotypes were subsequently challenged with a phloem-feeding insect (aphids: Myzus persicae), an insect herbivore (leafminers: Liriomyza trifolii), and two different necrotrophic fungal pathogens (Botrytis cinerea and Alternaria brassicicola). We also characterized the volatiles emitted by these plants upon aphid infestation or mechanical wounding and identified hexenyl acetate as the predominant compound in these volatile blends. Subsequently, we examined the signaling role of this compound in attracting the parasitoid wasp (Aphidius colemani), a natural enemy of aphids. PRINCIPAL FINDINGS: This study conclusively establishes that jasmonates and C(6)-aldehydes play distinct roles in plant defense responses. The jasmonates are indispensable metabolites in mediating the activation of direct plant-defense responses, whereas the C(6)-aldehyes are not. On the other hand, hexenyl acetate, an acetylated C(6)-aldehyde, is the predominant wound-inducible volatile signal that mediates indirect defense responses by directing tritrophic (plant-herbivore-natural enemy) interactions. SIGNIFICANCE: The data suggest that jasmonates and hexenyl acetate play distinct roles in mediating direct and indirect plant-defense responses. The potential advantage of this "division of labor" is to ensure the most effective defense strategy that minimizes incurred damages at a reduced metabolic cost.

Interlinking showy traits: co-engineering of scent and colour biosynthesis in flowers.

The phenylpropanoid pathway gives rise to metabolites that determine floral colour and fragrance. These metabolites are one of the main means used by plants to attract pollinators, thereby ensuring plant survival. A lack of knowledge about factors regulating scent production has prevented the successful enhancement of volatile phenylpropanoid production in flowers. In this study, the Production of Anthocyanin Pigment1 (Pap1) Myb transcription factor from Arabidopsis thaliana, known to regulate the production of non-volatile phenylpropanoids, including anthocyanins, was stably introduced into Petunia hybrida. In addition to an increase in pigmentation, Pap1-transgenic petunia flowers demonstrated an increase of up to tenfold in the production of volatile phenylpropanoid/benzenoid compounds. The dramatic increase in volatile production corresponded to the native nocturnal rhythms of volatile production in petunia. The application of phenylalanine to Pap1-transgenic flowers led to an increase in the otherwise negligible levels of volatiles emitted during the day to nocturnal levels. On the basis of gene expression profiling and the levels of pathway intermediates, it is proposed that both increased metabolic flux and transcriptional activation of scent and colour genes underlie the enhancement of petunia flower colour and scent production by Pap1. The co-ordinated regulation of metabolic steps within or between pathways involved in vital plant functions, as shown here for two showy traits determining plant-pollinator interactions, provides a clear advantage for plant survival. The use of a regulatory factor that activates scent production creates a new biotechnological strategy for the metabolic architecture of fragrance, leading to the creation of novel genetic variability for breeding purposes.