We established a step-by-step, experimentguided metabolomics procedure, based on LC-ESI-MS analysis, to generate a detailed picture of the changing metabolic profiles during late berry development in the important Italian grapevine cultivar Corvina. We sampled berries from four developmental time points and three postharvest time points during the withering process, and used chromatograms of methanolic extracts to test the performance of the MetAlign and MZmine data mining programs. MZmine achieved a better resolution and therefore generated a more useful data matrix. Then both the quantitative performance of the analytical platform and the matrix effect were assessed, and the final dataset was investigated by multivariate data analysis. Our analysis confirmed the results of previous studies but also revealed some novel findings, including the prevalence of two specific flavonoids in unripe berries and important differences between the developmental profiles of flavones and flavanones, suggesting that specific individual metabolites could have different functions, and that flavones and flavanones probably play quite distinct biological roles. Moreover, the hypothesis-free multivariate analysis of subsets of the wide data matrix evidentiated the relationships between the various classes of metabolites, such as those between anthocyanins and hydroxycinnamic acids and between flavan-3-ols and anthocyanins.

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Cytometry and flow cytometry were used to study characteristics of fluorescence of the DNA‐DAPI complex in nuclei released from different fresh and formaldehyde‐fixed pea (Pisum sativum L. cv. Lincoln) tissues. The two methods of isolation are compared and discussed as well as their possible use for quantitative analysis of DNA in plant tissues. With fixed tissues it is possible to obtain a number of nuclei sufficient for the flow cytometric analysis, even using small amounts of plant tissue.

The phenylpropanoid and flavonoid families include thousands of specialized metabolites that influence a wide range of processes in plants, including seed dispersal, auxin transport, photoprotection, mechanical support and protection against insect herbivory. Such metabolites play a key role in the protection of plants against abiotic stress, in many cases through their well-known ability to inhibit the formation of reactive oxygen species (ROS). However, the precise role of specific phenylpropanoid and flavonoid molecules is unclear. We therefore investigated the role of specific anthocyanins (ACs) and other phenylpropanoids that accumulate in carrot cells cultivated in vitro, focusing on their supposed ability to protect cells from heat stress. First we characterized the effects of heat stress to identify quantifiable morphological traits as markers of heat stress susceptibility. We then fed the cultures with precursors to induce the targeted accumulation of specific compounds, and compared the impact of heat stress in these cultures and unfed controls. Data modeling based on projection to latent structures (PLS) regression revealed that metabolites containing coumaric or caffeic acid, including ACs, correlate with less heat damage. Further experiments suggested that one of the cellular targets damaged by heat stress and protected by these metabolites is the actin microfilament cytoskeleton.

Root meristems of Pisum sativum L. cv. Lincoln were pulsed with bromodeoxyuridine, and after partial DNA denaturation the incorporated precursor was detected with an indirect immunofluorescent method by means of commercial anti‐bromodeoxyuridine monoclonal antibody and fluorescein isothiocyanate‐labeled secondary antibody. The nuclei were counterstained with the DNA‐specific fluorochrome 4′, 6‐diamidino‐2‐phenylindole (DAPI). The labeling indices (percentage of labeled calls) determined on slides prepared with the same nuclear population were very similar and in strict accordance with autoradiographic data. When DAPI fluorescence of the nuclei was measured, the unlabeled nuclei coincided with the G1 and part of the G2 regions, whereas all the labeled nuclei were in the S or G2 region. The method is reliable and, in contrast to autoradiography, data can be collected rapidly and almost immediately after the pulse. Furthermore, labeling index and the distribution of DNA contents can be obtained from the same side.