The influence of drying and storage conditions on the volatilome and cannabinoid content of Cannabis sativa L. inflorescences.

The increasing interest in hemp and cannabis poses new questions about the influence of drying and storage conditions on the overall aroma and cannabinoids profile of these products. Cannabis inflorescences are subjected to drying shortly after harvest and then to storage in different containers. These steps may cause a process of rapid deterioration with consequent changes in precious secondary metabolite content, negatively impacting on the product quality and potency. In this context, in this work, the investigation of the effects of freeze vs tray drying and three storage conditions on the preservation of cannabis compounds has been performed. A multi-trait approach, combining both solid-phase microextraction (SPME) two-dimensional gas chromatography coupled to mass spectrometry (SPME-GC × GC-MS) and high-performance liquid chromatography (HPLC), is presented for the first time. This approach has permitted to obtain the detailed characterisation of the whole cannabis matrix in terms of volatile compounds and cannabinoids. Moreover, multivariate statistical analyses were performed on the obtained data, helping to show that freeze drying conditions is useful to preserve cannabinoid content, preventing decarboxylation of acid cannabinoids, but leads to a loss of volatile compounds which are responsible for the cannabis aroma. Furthermore, among storage conditions, storage in glass bottle seems more beneficial for the retention of the initial VOC profile compared to open to air dry tray and closed high-density polyethylene box. However, the glass bottle storage condition causes formation of neutral cannabinoids at the expenses of the highly priced acid forms. This work will contribute to help define optimal storage conditions useful to produce highly valuable and high-quality products.

Composition and nutritional values of fatty acids in marine organisms by one-step microwave-assisted extraction/derivatization and comprehensive two-dimensional gas chromatography -flame ionization detector.

This work reports the characterization of the lipidic fraction of seven species of marine organisms gathered along the shoreline of the Po Delta Park of Emilia-Romagna Region (Italy) and of the north Adriatic Sea. Two species of oysters (Crassostrea gigas and Ostrea edulis), two species of clams (Chamelea gallina and Ruditapes philippinarum), one species of mussel (Mytilus galloprovincialis), one species of macroalgae (Ulva rigida), and one species of spiny dogfish (Squalus acanthias) were analyzed to characterize their fatty acids profile and related nutritional value. The lipid fraction was simultaneously extracted and transesterified into fatty acid methyl esters (FAMEs) by using a recently developed one-step microwave-assisted extraction/derivatization (MAED) method. The obtained FAMEs extract was analyzed by a rapid comprehensive multidimensional gas chromatography (GC × GC) method (30 min). The system was equipped with a reverse set of columns (polar × non-polar) connected through a reversed fill/flush flow modulator. The GC × GC system was coupled with a flame-ionization detector (FID) for both qualitative and quantitative purposes. The MAED- GC × GC-FID methodology was suitable in the context of samples containing high percentages of omega-3 PUFA. A total of 82 FAMEs were tentatively identified using standards, literature data, and the two-dimensional plot location. FAME profiles obtained with the proposed approach were comparable with reference methods (AOCS Ce 2b-11), showing no significant differences. Moreover, to determine the food nutritional value of the samples investigated, the most common nutritional indices (index of atherogenicity, index thrombogenicity, hypocholesterolemic/hypercholesterolemic ratio, health-promoting index, unsaturation index, and the fish lipid quality index) were calculated from FAME profiles. Among the samples investigated, Squalus acanthias presented the best nutritional score, while Ruditapes philippinarum had the worst score in 3 out of 6 indices.

Vacuum-assisted and multi-cumulative trapping in headspace solid-phase microextraction combined with comprehensive multidimensional chromatography-mass spectrometry for profiling virgin olive oil aroma.

In the present work vacuum (Vac) and multiple cumulative trapping (MCT) headspace solid phase microextraction (HS-SPME) were evaluated as alternative or combined techniques for the volatile profiling. A higher extraction performance for semi-volatiles was shown by all three techniques. Synergic combination of Vac and MCT showed up to 5-times extraction power for less volatile compounds. The hyphenation of said techniques with comprehensive two-dimensional gas chromatography (GC × GC) enabled a comprehensive analysis of the volatilome. Firstly, 18 targeted quality markers, previously defined by means of classical HS-SPME, were explored for their ability to classify commercial categories. The applicability of such markers proved to be limited with the alternative sampling techniques. An untargeted approach enables the selection of specific features for each technique showing a better classification capacity of the commercial categories. No misclassifications were observed, except for one extra virgin olive oil classified as virgin olive oil in 3 × 10 min Vac-MCT-HS-SPME.

Biochemical and molecular changes in peach fruit exposed to cold stress conditions.

Storage or transportation temperature is very important for preserving the quality of fruit. However, low temperature in sensitive fruit such as peach can induce loss of quality. Fruit exposed to a specific range of temperatures and for a longer period can show chilling injury (CI) symptoms. The susceptibility to CI at low temperature varies among cultivars and genetic backgrounds. Along with agronomic management, appropriate postharvest management can limit quality losses. The importance of correct temperature management during postharvest handling has been widely demonstrated. Nowadays, due to long-distance markets and complex logistics that require multiple actors, the management of storage/transportation conditions is crucial for the quality of products reaching the consumer.Peach fruit exposed to low temperatures activate a suite of physiological, metabolomic, and molecular changes that attempt to counteract the negative effects of chilling stress. In this review an overview of the factors involved, and plant responses is presented and critically discussed. Physiological disorders associated with CI generally only appear after the storage/transportation, hence early detection methods are needed to monitor quality and detect internal changes which will lead to CI development. CI detection tools are assessed: they need to be easy to use, and preferably non-destructive to avoid loss of products.

Molecular Mechanisms Underlying Potential Pathogen Resistance in Cannabis sativa.

Cannabis (Cannabis sativa L.) is one of the earliest cultivated crops, valued for producing a broad spectrum of compounds used in medicinal products and being a source of food and fibre. Despite the availability of its genome sequences, few studies explore the molecular mechanisms involved in pathogen defense, and the underlying biological pathways are poorly defined in places. Here, we provide an overview of Cannabis defence responses against common pathogens, such as Golovinomyces spp., Fusarium spp., Botrytis cinerea and Pythium spp. For each of these pathogens, after a summary of their characteristics and symptoms, we explore studies identifying genes involved in Cannabis resistance mechanisms. Many studies focus on the potential involvement of disease-resistance genes, while others refer to other plants however whose results may be of use for Cannabis research. Omics investigations allowing the identification of candidate defence genes are highlighted, and genome editing approaches to generate resistant Cannabis species based on CRISPR/Cas9 technology are discussed. According to the emerging results, a potential defence model including both immune and defence mechanisms in Cannabis plant-pathogen interactions is finally proposed. To our knowledge, this is the first review of the molecular mechanisms underlying pathogen resistance in Cannabis.

Storage of halved strawberry fruits affects aroma, phytochemical content and gene expression, and is affected by pre-harvest factors.

Introduction: Strawberry fruit are highly valued for their aroma which develops during ripening. However, they have a short shelf-life. Low temperature storage is routinely used to extend shelf-life for transport and storage in the supply chain, however cold storage can also affect fruit aroma. Some fruit continue to ripen during chilled storage; however, strawberries are a non-climacteric fruit and hence ripening postharvest is limited. Although most strawberry fruit is sold whole, halved fruit is also used in ready to eat fresh fruit salads which are of increasing consumer demand and pose additional challenges to fresh fruit storage. Methods: To better understand the effects of cold storage, volatilomic and transcriptomic analyses were applied to halved Fragaria x ananassa cv. Elsanta fruit stored at 4 or 8°C for up to 12 days over two growing seasons. Results and discussion: The volatile organic compound (VOC) profile differed between 4 or 8°C on most days of storage. Major differences were detected between the two different years of harvest indicating that aroma change at harvest and during storage is highly dependent on environmental factors during growth. The major component of the aroma profile in both years was esters. Over 3000 genes changed in expression over 5 days of storage at 8°C in transcriptome analysis. Overall, phenylpropanoid metabolism, which may also affect VOCs, and starch metabolism were the most significantly affected pathways. Genes involved in autophagy were also differentially expressed. Expression of genes from 43 different transcription factor (TF) families changed in expression: mostly they were down-regulated but NAC and WRKY family genes were mainly up-regulated. Given the high ester representation amongst VOCs, the down-regulation of an alcohol acyl transferase (AAT) during storage is significant. A total of 113 differentially expressed genes were co-regulated with the AAT gene, including seven TFs. These may be potential AAT regulators.

Exploring different high-capacity tools and extraction modes to characterize the aroma of brewed coffee.

In the present work, the potential benefit of using multi-cumulative trapping headspace extraction was explored by comparing the results using solid-phase microextraction (SPME) coated with divinylbenzene/carboxen/polydimethylsiloxane and a probe-like tool coated with polydimethylsiloxane. The efficiency of a single 30-min extraction, already explored in previous work, was compared with that of multiple shorter extractions. We evaluated three different conditions, i.e., three repeated extractions for 10 min each from different sample vials (for both the probe-like tool and SPME) or from the same vial (for SPME) containing brewed coffee. The entire study was performed using comprehensive two-dimensional gas chromatography coupled with mass spectrometry. The two-dimensional plots were aligned and integrated using a tile-sum approach before any statistical analysis. A detailed comparison of all the tested conditions was performed on a set of 25 targeted compounds. Although a single 30-min extraction using the probe-like tool provided a significantly higher compound intensity than SPME single extraction, the use of multiple shorter extractions with SPME showed similar results. However, multiple extractions with the probe-like tool showed a greater increase in the number of extracted compounds. Furthermore, an untargeted cross-sample comparison was performed to evaluate the ability of the two tested tools and the different extraction procedures in differentiating between espresso-brewed coffee samples obtained from capsules made of different packaging materials (i.e., compostable capsules, aluminum capsules, aluminum multilayer pack). The highest explained variance was obtained using the probe-like tool and multiple extractions (91.6% compared to 83.9% of the single extraction); nevertheless, SPME multiple extractions showed similar results with 88.3% of variance explained.

Comprehensive analysis of floral scent and fatty acids in nectar of Silene nutans through modern analytical gas chromatography techniques.

The aim of this work was to show the potential of multidimensional gas chromatography combined with mass spectrometry and suitable chemometrics means based on untargeted and profiling data analysis to strengthen the information provided by floral scent and nectar fatty acids of four genetically differentiated lineages (E1, W1, W2, and W3) of the nocturnal moth-pollinated herb Silene nutans. Volatile organic compounds emitted by flowers were trapped for a total of 42 samples by in-vivo sampling dynamic head space for analysing floral scent by untargeted approach, while 37 samples of nectar were collected for analysing fatty acids through profiling analysis. The resulting data from floral scent analysis were aligned and compared using a tile-based methodology followed by data mining to access high-level information. Based on floral scent and nectar fatty acid results, it was possible to distinguish E1 from the W lineages, and W3 from W1 and W2. This work puts the bases for a larger study aiming to clarify the existence of prezygotic barriers involved in speciation among lineages of S. nutans, and thus the possible implication of different flower scents and nectar compositions in this phenomenon.

Unraveling the impact of the capsule material on the aroma of brewed coffee by headspace analysis using a HiSorb probe followed by reverse fill/flush flow modulation GC×GC-MS.

The present paper discusses the use of a high-concentration-capacity tool, HiSorb, to investigate the impact of capsule material on the aroma profile of espresso-brewed coffee. The specific high-concentration-capacity probe used is characterized by a sorbent volume (63 µL) intermediate between the solid-phase microextraction (SPME) fiber (0.6 µL) and the stir-bar sorptive extraction rod (126 µL). The extraction performance of the HiSorb was compared, in terms of both absolute signal and compound coverage, with both an equivalent sorbent (polydimethylsiloxane) and a divinylbenzene/carboxen/polydimethylsiloxane SPME fiber using both targeted and untargeted approaches. The HiSorb showed superior extraction compared with the SPME fibers. The HiSorb was then optimized in terms of extraction time and temperature and used to investigate the volatile profile of 23 espresso-brewed coffees prepared with capsules made of different materials-aluminum, compostable, and aluminum multilayer pack-prepared using a refillable capsule. Comprehensive two-dimensional gas chromatography equipped with a reverse fill/flush flow modulator and coupled to mass spectrometry was used to obtain a chromatographic fingerprint of the volatile profile of the brewed coffee. The data were aligned and compared using a tile-based approach, and the results were obtained by performing raw data mining within the same software platform. The data mining enabled the extraction of informative features responsible for the differentiation between the different capsule materials, showing a significant depletion in aroma intensity in the compostable capsule.

Comparative transcriptomic profiling of peach and nectarine cultivars reveals cultivar-specific responses to chilled postharvest storage.

Introduction: Peach (Prunus persica (L.) Batsch,) and nectarine fruits (Prunus persica (L.) Batsch, var nectarine), are characterized by a rapid deterioration at room temperature. Therefore, cold storage is widely used to delay fruit post-harvest ripening and extend fruit commercial life. Physiological disorders, collectively known as chilling injury, can develop typically after 3 weeks of low-temperature storage and affect fruit quality. Methods: A comparative transcriptomic analysis was performed to identify regulatory pathways that develop before chilling injury symptoms are detectable using next generation sequencing on the fruits of two contrasting cultivars, one peach (Sagittaria) and one nectarine, (Big Top), over 14 days of postharvest cold storage. Results: There was a progressive increase in the number of differentially expressed genes between time points (DEGs) in both cultivars. More (1264) time point DEGs were identified in 'Big Top' compared to 'Sagittaria' (746 DEGs). Both cultivars showed a downregulation of pathways related to photosynthesis, and an upregulation of pathways related to amino sugars, nucleotide sugar metabolism and plant hormone signal transduction with ethylene pathways being most affected. Expression patterns of ethylene related genes (including biosynthesis, signaling and ERF transcription factors) correlated with genes involved in cell wall modification, membrane composition, pathogen and stress response, which are all involved later during storage in development of chilling injury. Discussion: Overall, the results show that common pathways are activated in the fruit of 'Big Top' nectarine and 'Sagittaria' peach in response to cold storage but include also differences that are cultivar-specific responses.

Fruitomics: The Importance of Combining Sensory and Chemical Analyses in Assessing Cold Storage Responses of Six Peach (Prunus persica L. Batsch) Cultivars.

Cold storage is used to extend peach commercial life, but can affect quality. Quality changes are assessed through the content of nutritionally relevant compounds, aroma, physical characters and/or sensorially. Here, six peach and nectarine cultivars were sampled at commercial harvest and after 7 days of 1 °C storage. A trained panel was used to evaluate sensorial characters, while carotenoids, phenolics, vitamin C, total sugars, and qualitative traits including firmness, titrable acidity and soluble solid content were integrated with volatile organic compound (VOC) analysis previously reported. The different analyses reveal interesting patterns of correlation, and the six cultivars responded differently to cold storage. Sensory parameters were correlated with 64 VOCs and seven intrinsic characters. Acidity, firmness, and 10 VOCs were strongly negatively correlated with harmony and sweetness, but positively correlated with bitterness, astringency, and crunchiness. In contrast, Brix, b-carotene, and six VOCs were positively correlated with harmony and sweetness.

Multi-Omics Approaches to Study Molecular Mechanisms in Cannabis sativa.

Cannabis (Cannabis sativa L.), also known as hemp, is one of the oldest cultivated crops, grown for both its use in textile and cordage production, and its unique chemical properties. However, due to the legislation regulating cannabis cultivation, it is not a well characterized crop, especially regarding molecular and genetic pathways. Only recently have regulations begun to ease enough to allow more widespread cannabis research, which, coupled with the availability of cannabis genome sequences, is fuelling the interest of the scientific community. In this review, we provide a summary of cannabis molecular resources focusing on the most recent and relevant genomics, transcriptomics and metabolomics approaches and investigations. Multi-omics methods are discussed, with this combined approach being a powerful tool to identify correlations between biological processes and metabolic pathways across diverse omics layers, and to better elucidate the relationships between cannabis sub-species. The correlations between genotypes and phenotypes, as well as novel metabolites with therapeutic potential are also explored in the context of cannabis breeding programs. However, further studies are needed to fully elucidate the complex metabolomic matrix of this crop. For this reason, some key points for future research activities are discussed, relying on multi-omics approaches.

Cross-Cultural Differences between Italian and UK Consumer Preferences for 'Big Top' Nectarines in Relation to Cold Storage.

Nectarines are perishable fruits grown in Southern Europe, valued for their sensorial properties. Chilling is used in the supply chain for Northern European consumers, while Southern European consumers can access fresh, locally grown fruit or cold-stored supermarket fruit. Cold storage and fruit ripening affect texture and flavour. Here a consumer survey and hedonic testing compared the appreciation of nectarines (cv. Big Top) in Italy and at two UK sites (n = 359). Fruit was at the commercial harvest stage, or stored at 1 °C or 5 °C for seven days, then sampled after two days' (Italy and one UK site) or four days' (second UK site) ambient recovery. In the consumer survey, the most important factors involved in purchase decision were ripeness, texture, colour, taste and price. Named varieties were more important to Italian than UK respondents, whilst ripeness, price, taste, blemishes, aroma, and 'best before date' were more important in the UK. In sensory analyses, fruits at the commercial harvest stage were preferred to those stored at 1 °C. Preference for the 5 °C stored peaches depended on recovery time. Distinct clusters of peach sensorial attributes were positively or negatively linked to hedonic rating. Factors important in purchase decisions did not affect hedonic rating in the tasting.

Identifying volatile and non-volatile organic compounds to discriminate cultivar, growth location, and stage of ripening in olive fruits and oils.

BACKGROUND: There is increasing consumer demand for olive oil to be traceable. However, genotype, environmental factors, and stage of maturity, all affect the flavor and composition of both the olives and olive oil. Few studies have included all three variables. Key metabolites include lipids, phenolics, and a wide range of volatile organic compounds (VOCs), which provide the olives and oil with their characteristic flavor. Here we aim to identify markers that are able to discriminate between cultivars, that can identify growth location, and can discriminate stages of fruit maturity. 'Nocellara messinese' and 'Carolea' olive fruits were grown at three locations differing in altitude in Calabria, Italy, and harvested at three stages of maturity. Oil was analyzed from the two most mature stages. RESULTS: Nine and 20 characters discriminated all fruit and oil samples respectively, and relative abundance of two fatty acids distinguished all oils. Whole VOC profiles discriminated among the least mature olives, and oil VOC profiles discriminated location and cultivar at both stages. Three VOCs putatively identified as hexanal, methyl acetate, and 3-hexen-1-ol differentiated all samples of oils from the most mature fruit stage. CONCLUSION: The results confirm that interactions of location, cultivar and fruit maturity stage are critical for the overall pattern of aroma compounds, and identify potential markers of commercial relevance. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Exploring multiple-cumulative trapping solid-phase microextraction coupled to gas chromatography-mass spectrometry for quality and authenticity assessment of olive oil.

This study explores the potential of an innovative multi-cumulative trapping headspace solid-phase microextraction approach coupled with untargeted data analysis to enhance the information provided by aroma profiling of virgin olive oil. Sixty-nine samples of different olive oil commercial categories (extra-virgin, virgin and lampante oil) and different geographical origins were analysed using this novel workflow. The results from each sample were aligned and compared using for the first time a tile-based approach to enable the mining of all of the raw data within the chemometrics platform without any pre-processing methods. The data matrix obtained allowed the extraction of multiple-level information from the volatile profile of the samples. Not only was it possible to classify the samples within the commercial category that they belonged to, but the same data also provided interesting information regarding the geographical origin of the extra-virgin olive oil.

Mutation of Arabidopsis Copper-Containing Amine Oxidase Gene AtCuAOδ Alters Polyamines, Reduces Gibberellin Content and Affects Development.

Polyamines (PAs) are essential metabolites in plants performing multiple functions during growth and development. Copper-containing amine oxidases (CuAOs) catalyse the catabolism of PAs and in Arabidopsis thaliana are encoded by a gene family. Two mutants of one gene family member, AtCuAOδ, showed delayed seed germination, leaf emergence, and flowering time. The height of the primary inflorescence shoot was reduced, and developmental leaf senescence was delayed. Siliques were significantly longer in mutant lines and contained more seeds. The phenotype of AtCuAOδ over-expressors was less affected. Before flowering, there was a significant increase in putrescine in AtCuAOδ mutant leaves compared to wild type (WT), while after flowering both spermidine and spermine concentrations were significantly higher than in WT leaves. The expression of GA (gibberellic acid) biosynthetic genes was repressed and the content of GA1, GA7, GA8, GA9, and GA20 was reduced in the mutants. The inhibitor of copper-containing amine oxidases, aminoguanidine hydrochloride, mimicked the effect of AtCuAOδ mutation on WT seed germination. Delayed germination, reduced shoot height, and delayed flowering in the mutants were rescued by GA3 treatment. These data strongly suggest AtCuAOδ is an important gene regulating PA homeostasis, and that a perturbation of PAs affects plant development through a reduction in GA biosynthesis.

Fruit volatilome profiling through GC × GC-ToF-MS and gene expression analyses reveal differences amongst peach cultivars in their response to cold storage.

Peaches have a short shelf life and require chilling during storage and transport. Peach aroma is important for consumer preference and determined by underlying metabolic pathways and gene expression. Differences in aroma (profiles of volatile organic compounds, VOCs) have been widely reported across cultivars and in response to cold storage. However, few studies used intact peaches, or used equilibrium sampling methods subject to saturation. We analysed VOC profiles using TD-GC × GC-ToF-MS and expression of 12 key VOC pathway genes of intact fruit from six cultivars (three peaches, three nectarines) before and after storage at 1 °C for 7 days including 36 h shelf life storage at 20 °C. Two dimensional GC (GC × GC) significantly enhances discrimination of thermal desorption gas chromatography time-of-flight mass spectrometry (TD-GC-ToF-MS) and detected a total of 115 VOCs. A subset of 15 VOCs from analysis with Random Forest discriminated between cultivars. Another 16 VOCs correlated strongly with expression profiles of eleven key genes in the lipoxygenase pathway, and both expression profiles and VOCs discriminated amongst cultivars, peach versus nectarines and between treatments. The cultivar-specific response to cold storage underlines the need to understand more fully the genetic basis for VOC changes across cultivars.

Comprehensive Two-Dimensional Gas Chromatography with Mass Spectrometry: Toward a Super-Resolved Separation Technique.

A data interpretation and processing approach for improved compound identification and data presentation in comprehensive two-dimensional gas chromatography (GC×GC) is described. A footprint peak of a compound in 2D space can be represented by a centroid or peak apex, similar to the data-reduced histogram spectra used in mass spectrometry. The workflow was demonstrated on data from GC×GC-TOFMS. Peaks in a modulated chromatogram were initially detected by conventional chromatographic integration, followed by a curve-fitting approach, which interpolated high-precision, absolute retention times for all modulated peaks. First dimension retention time (1tR) was obtained by using an exponentially modified Gaussian (EMG) fitting model for near-Gaussian distributed subpeaks, polynomial fitting for highly asymmetrical peaks, and parabolic fitting for under-sampled peaks, which allows determination of a precise 1tR, considering the dwell-time arising from modulation and 2tR. Area summation of the modulated peaks belonging to the same compound was then performed to yield the total peak area. Each compound in the GC×GC-MS result was then represented by its position at the intersecting coordinates, (1tR, 2tR), in the 2D separation plane, having a height of the same magnitude as the total component summed area. This results in a novel and uncluttered GC×GC output convention based on the scripted total ion chromatogram (TIC) data with precise 1tR, 2tR, and area. Comparison between the contour plots from the scripted and conventional TIC revealed improved data presentation, accompanied by an apparent enhanced resolution. The described approach was applied to the identification of 177 aroma compounds from peaches as indicators of fruit quality.

Short-Term Post-Harvest Stress that Affects Profiles of Volatile Organic Compounds and Gene Expression in Rocket Salad During Early Post-Harvest Senescence.

Once harvested, leaves undergo a process of senescence which shares some features with developmental senescence. These include changes in gene expression, metabolites, and loss of photosynthetic capacity. Of particular interest in fresh produce are changes in nutrient content and the aroma, which is dependent on the profile of volatile organic compounds (VOCs). Leafy salads are subjected to multiple stresses during and shortly after harvest, including mechanical damage, storage or transport under different temperature regimes, and low light. These are thought to impact on later shelf life performance by altering the progress of post-harvest senescence. Short term stresses in the first 24 h after harvest were simulated in wild rocket (Diplotaxis tenuifolia). These included dark (ambient temperature), dark and wounding (ambient temperature), and storage at 4 °C in darkness. The effects of stresses were monitored immediately afterwards and after one week of storage at 10 °C. Expression changes in two NAC transcription factors (orthologues of ANAC059 and ANAC019), and a gene involved in isothiocyanate production (thiocyanate methyltransferase, TMT) were evident immediately after stress treatments with some expression changes persisting following storage. Vitamin C loss and microbial growth on leaves were also affected by stress treatments. VOC profiles were differentially affected by stress treatments and the storage period. Overall, short term post-harvest stresses affected multiple aspects of rocket leaf senescence during chilled storage even after a week. However, different stress combinations elicited different responses.

Expression of Arabidopsis WEE1 in tobacco induces unexpected morphological and developmental changes.

WEE1 regulates the cell cycle by inactivating cyclin dependent protein kinases (CDKs) via phosphorylation. In yeast and animal cells, CDC25 phosphatase dephosphorylates the CDK releasing cells into mitosis, but in plants, its role is less clear. Expression of fission yeast CDC25 (Spcdc25) in tobacco results in small cell size, premature flowering and increased shoot morphogenetic capacity in culture. When Arath;WEE1 is over-expressed in Arabidopsis, root apical meristem cell size increases, and morphogenetic capacity of cultured hypocotyls is reduced. However expression of Arath;WEE1 in tobacco plants resulted in precocious flowering and increased shoot morphogenesis of stem explants, and in BY2 cultures cell size was reduced. This phenotype is similar to expression of Spcdc25 and is consistent with a dominant negative effect on WEE1 action. Consistent with this putative mechanism, WEE1 protein levels fell and CDKB levels rose prematurely, coinciding with early mitosis. The phenotype is not due to sense-mediated silencing of WEE1, as overall levels of WEE1 transcript were not reduced in BY2 lines expressing Arath;WEE1. However the pattern of native WEE1 transcript accumulation through the cell cycle was altered by Arath;WEE1 expression, suggesting feedback inhibition of native WEE1 transcription.