Gene expression analysis of rocket salad under pre-harvest and postharvest stresses : a transcriptomic resource for Diplotaxis tenuifolia

Diplotaxis tenuifolia L. is of important economic value in the fresh-cut industry for its nutraceutical and sensorial properties. However, information on the molecular mechanisms conferring tolerance of harvested leaves to pre- and postharvest stresses during processing and shelf-life have never been investigated. Here, we provide the first transcriptomic resource of rocket by de novo RNA sequencing assembly, functional annotation and stress-induced expression analysis of 33874 transcripts. Transcriptomic changes in leaves subjected to commercially-relevant pre-harvest (salinity, heat and nitrogen starvation) and postharvest stresses (cold, dehydration, dark, wounding) known to affect quality and shelf-life were analysed 24h after stress treatment, a timing relevant to subsequent processing of salad leaves. Transcription factors and genes involved in plant growth regulator signaling, autophagy, senescence and glucosinolate metabolism were the most affected by the stresses. Hundreds of genes with unknown function but uniquely expressed under stress were identified, providing candidates to investigate stress responses in rocket. Dehydration and wounding had the greatest effect on the transcriptome and different stresses elicited changes in the expression of genes related to overlapping groups of hormones. These data will allow development of approaches targeted at improving stress tolerance, quality and shelf-life of rocket with direct applications in the fresh-cut industries

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 \ub0C in darkness. The effects of stresses were monitored immediately afterwards and after one week of storage at 10 \ub0C. 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

Effect of CDC25 and WEE1 on Plant Cell Cycle and Morphogenesis

The cell cycle comprise the four phases of, G1, S-phase, G2 and mitosis. Two critical transitions are G1/S and G2/M; the latter is regulated by WEE1 kinase and CDC25 phosphatases. The scope of this thesis was to investigate the regulation of the G2/M transition of the cell cycle by WEE1 and CDC25, and how these genes interface with plant growth regulators in Arabidopsis thaliana. In Arabidopsis roots, the frequency of lateral roots was found to be increased by ectopic expression of Schizosaccharomyces pombe (Sp)cdc25e and reduced by Arath;WEE1 expression. I examined the effect of Arath;WEE1 and Spcdc25 on induction of shoots and roots in Arabidopsis hypocotyls in vitro. Hypocotyl explants from two over-expressing WEE1 lines , three T-DNA insertion lines and two expressing cdc25 (Spcdc25e) lines together with wild type (WT) were cultured on two-way gradients of kinetin (Kin) and naphthyl acetic acid (NAA). Below a threshold concentration of NAA (100 ng ml-1), WEE1 repressed morphogenesis in vitro, whereas at all NAA/Kin combinations Spcdc25 promoted morphogenesis (particularly root formation) over and above that in WT. Loss of function wee1-1 cultures were very similar to WT. Quantitative data indicated a significant increase in the frequency of root formation in Spcdc25e cultures compared with WT particularly at low Kin concentrations, and WEE1oe’s repressive effect was overcome by NAA but not Kin. In conclusion, WEE1 has a repressive effect on morphogenesis in vitro that can be overcome by auxin whereas Spcd25 by-passes a cytokinin requirement for the induction of morphogenesis in vitro. The role of CDC25 and WEE1 in DNA damage responses was also analysed. Two over-expressing Arath;CDC25 lines and T-DNA mutants showed no difference to WT either in standard conditions or zeocin-supplemented treatments. However, root length was longer in Arath;CDC25oe lines treated with hydroxyurea (HU) and lateral root number was increased compared to WT. This suggests a differential response of Arath;CDC25oe in the DNA replication (HU-induced) and DNA damage (zeocin-induced) checkpoints (Chapter 5). Finally the roles of WEE1 and CDC25 in cell cycle regulation were examined using tobacco TBY-2 cell cultures expressing Arath;WEE1, Nicotiana tabacum (Nicta)WEE1 or Arath;CDC25. Whilst Nicta;WEE1 lengthened G2 of the cell cycle, Arath;WEE1 had an unusual effect of shortening G2 phase and Arath;CDC25 had no observable effect (Chapter 6)

Wounding tomato fruit elicits ripening-stage specific changes in gene expression and production of volatile compounds

Fleshy fruits develop from an unripe organ that needs to be protected from damage to a ripe organ that attracts frugivores for seed dispersal through production of volatile organic compounds (VOCs). Thus, different responses to wounding damage are predicted. The aim of this study was to discover whether wound-induced changes in the transcriptome and VOC production alter as tomato transitions from unripe to ripe. Transcript changes were analysed 3h post-wounding using microarray analysis in two commercial salad-tomato (Solanum lycopersicum L.) cultivars: Luna Rossa and AVG, chosen for their high aroma production. This was followed by quantitative PCR on Luna Rossa genes involved in VOC biosynthesis and defence responses. VOCs elicited by wounding at different ripening stages were analysed by solid phase micro extraction and gas chromatography-mass spectrometry. Approximately 4000 differentially expressed genes were identified in the cultivar AVG and 2500 in Luna Rossa. In both cultivars the majority of genes were up-regulated and the most affected pathways were metabolism of terpenes, carotenoids, and lipids. Defence-related genes were mostly up-regulated in immature stages of development, whereas expression of genes related to VOCs changed at riper stages. More than 40 VOCs were detected and profiles changed with ripening stage. Thus, both transcriptome and VOC profiles elicited by wounding depend on stage of ripening, indicating a shift from defence to attraction

Multitrait Analysis of Fresh-Cut Cantaloupe Melon Enables Discrimination Between Storage Times and Temperatures and Identifies Potential Markers for Quality Assessments

Fresh-cut cantaloupe melon is valued for its aroma but is highly perishable. Temperature of storage (typically 0-5°C) is critical for maintaining fresh-cut melon quality, but often reaches 10 °C during transportation and in retail outlets. A comparison amongst 0, 5 and 10 °C storage temperatures for fresh-cut melon over 14 days reveals that storage at 0 °C is optimal for avoiding increases in microbial load and loss of vitamin C especially at later time points. However, higher temperatures maintain better the balance of esters (acetate versus non-acetate) and phenolic content. The whole volatile organic compound (VOC) profile can be used to discriminate both time and temperature effects especially at earlier time points. Potential VOC markers for changes in vitamin C from day 0 to day 6 of storage (3-methyl butane nitrile) and temperature (limonene) are identified through a multi-trait analysis

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 7 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 \ub0C for 7 days including 36 h shelf life storage at 20 \ub0C. Two dimensional GC (GC 7 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

Tailed Forisomes of Canavalia gladiata: A New Model to Study Ca2+-driven Protein Contractility

Background and Aims Forisomes are Ca2+-dependent contractile protein bodies that form reversible plugs in sieve tubes of faboid legumes. Previous work employed Vicia faba forisomes, a not entirely unproblematic experimental system. The aim of this study was to seek to establish a superior model to study these intriguing actuators. Methods Existing isolation procedures were modified to study the exceptionally large, tailed forisomes of Canavalia gladiata by differential interference contrast microscopy in vitro. To analyse contraction/expansion kinetics quantitatively, a geometric model was devised which enabled the computation of time-courses of derived parameters such as forisome volume from simple parameters readily determined on micrographs. Key Results Advantages of C. gladiata over previously utilized species include the enormous size of its forisomes (up to 55 µm long), the presence of tails which facilitate micromanipulation of individual forisomes, and the possibility of collecting material repeatedly from these fast-growing vines without sacrificing the plants. The main bodies of isolated Canavalia forisomes were box-shaped with square cross-sections and basically retained this shape in all stages of contraction. Ca2+-induced a 6-fold volume increase within about 10-15 s; the reverse reaction following Ca2+-depletion proceeded in a fraction of that time. Conclusions The sword bean C. gladiata provides a superior experimental system which will prove indispensable in physiological, biophysical, ultrastructural and molecular studies on the unique ATP-independent contractility of forisomes