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.

No abstract

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.

Plant cells are endowed with two distinct DNA polymerases [ 1,2] whose properties closely resemble those of the DNA polymerases cr and y present in animal cells [3,4]. The plant DNA polymerases have consequently been named o-like [ 1 ] and y-like [2].The B-like DNA polymerase activity is the most abundant in cultured plant cells [l ] and responds to changes in the rate of cell multiplication, whereas experiments with spinach leaves have shown that the y-like DNA polymerase is present in the chloroplast 121.A DNA polymerase has also been isolated from the mitochondria of wheat embryos [S]. Spinach mitochondria may also contain a DNA polymerase whose properties are partially different from those of the y-like DNA polymerase isolated from the chloroplasts of the same cells and are similar to those of the wheat embryo enzyme (unpublished).However, no evidence is available as yet on the existence in plant cells of a DNA repair enzyme similar to the DNA polymerase p of mammalian cellsBy analogy with animal cells, the assignment of functions to the DNA polymerases in plant cells is hampered by the lack of conditional mutants defective in DNA synthesis. Thus, we approach this problem by exploiting the properties of aphidicolin and of ethidium bromide.Aphidicolin We now describe the effect of aphidicolin and of ethidium bromide on the synthesis of DNA in the nucleus, c~oroplast and mitochondrion of cultured rice cells, as assessed by autoradiography at light and electron microscopy. The results show that aphidicolin specifically prevents the synthesis of nuclear DNA, while it has no effect on the synthesis of the organellar DNA, the latter being specifically affected only by ethidium bromide. This, together with the previous demonstration that only the a-like DNA polymerase is inhibited in vitro by aphidicolin [8], proves that the plant a-like DNA polymerase plays an essential role in the replication of nuclear DNA, and that this enzyme is not involved in the replication of plastid and mitochondriai DNA. Thus, the replication of the plant organellar DNA requires different DNA polymerases, the aphidicolin-resistant DNA polymerases present in the chloroplast [2] and in the plant mitochondrion [S J being the best candidates for this function.

No abstract

Dehydrin-like proteins have been detected in nuclei and cytoplasm of meristematic root tip cells from pea seedlings subjected to slow dehydration at 90 % relative humidity for 48 h or more. Evidence was gained from Western blotting and immunocytochemical experiments using an antibody raised against the conserved domain of dehydrin proteins. Flow cytometer analysis has shown that cycling cells of root tip meristems from dehydrated seedlings are mostly arrested in G2 phase. Other stress treatments thought to involve water depletion (osmotic stress, cold treatment) or to modulate cell response to water deficit (abscisic acid) gave less clear-cut results with all treatments lowering the proportion of cells entering the S phase, but without a definite and persistent arrest in any preferential phase of the cycle. Possible interrelationships between G2 arrest and dehydrin production are discussed.# 1997 Annals of Botany Company