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.

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.

Cadmium and/or copper excess induce interdependent metal accumulation, DNA methylation, induction of metal chelators and antioxidant defences in the seagrass Zostera marina.

In this investigation, we assessed the effects of Cu and/or Cd excess on physiological and metabolic processes of the widespread seagrass Zostera marina. Adult were exposed to low Cd and Cu (0.89 and 0.8 µM, respectively) and high Cd and Cu (8.9 and 2.4 µM, respectively) for 6 d at: Control conditions; low Cu; high Cu; low Cd; high Cd; low Cd and low Cu; and high Cd and high Cu. Photosynthetic performance decreased under single and combined treatments, although effects were more negative under Cu than Cd. Total Cu accumulation was higher than Cd, under single and combined treatments; however, their accumulation was generally lower when applied together, suggesting competition among them. Levels of glutathione (GSH) and phytochelatins (PCs) followed patterns similar to metal accumulation, with up to PC5, displaying adaptations in tolerance. A metallothionein (MET) gene showed upregulation only at high Cd, low Cu, and high Cu. The expression of the enzymes glutathione reductase (GR), ascorbate peroxidase (APX), and catalase (CAT) was greatest at high Cu, and at high Cd and Cu together; the highest expression was under Cu, alone and combined. Both metals induced upregulation of the DNA methyltransferases CMT3 and DRM2, with the highest expression at single Cu. The DNA demethylation ROS1 was overexpressed in treatments containing high Cu, suggesting epigenetic modifications. The results show that under copper and/or cadmium, Z. marina was still biologically viable; certainly based, at least in part, on the induction of metal chelators, antioxidant defences and methylation/demethylation pathways of gene regulation.

Plant WEE1 kinase is cell cycle regulated and removed at mitosis via the 26S proteasome machinery.

In yeasts and animals, premature entry into mitosis is prevented by the inhibitory phosphorylation of cyclin-dependent kinase (CDK) by WEE1 kinase, and, at mitosis, WEE1 protein is removed through the action of the 26S proteasome. Although in higher plants WEE1 function has been confirmed in the DNA replication checkpoint, Arabidopsis wee1 insertion mutants grow normally, and a role for the protein in the G2/M transition during an unperturbed plant cell cycle is yet to be confirmed. Here data are presented showing that the inhibitory effect of WEE1 on CDK activity in tobacco BY-2 cell cultures is cell cycle regulated independently of the DNA replication checkpoint: it is high during S-phase but drops as cells traverse G2 and enter mitosis. To investigate this mechanism further, a yeast two-hybrid screen was undertaken to identify proteins interacting with Arabidopsis WEE1. Three F-box proteins and a subunit of the proteasome complex were identified, and bimolecular fluorescence complementation confirmed an interaction between AtWEE1 and the F-box protein SKP1 interacting partner 1 (SKIP1). Furthermore, the AtWEE1-green fluorescent protein (GFP) signal in Arabidopsis primary roots treated with the proteasome inhibitor MG132 was significantly increased compared with mock-treated controls. Expression of AtWEE1-YFP(C) (C-terminal portion of yellow fluorescent protein) or AtWEE1 per se in tobacco BY-2 cells resulted in a premature increase in the mitotic index compared with controls, whereas co-expression of AtSKIP1-YFP(N) negated this effect. These data support a role for WEE1 in a normal plant cell cycle and its removal at mitosis via the 26S proteasome.

Gene dosage effect of WEE1 on growth and morphogenesis from arabidopsis hypocotyl explants.

BACKGROUND AND AIMS: How plant cell-cycle genes interface with development is unclear. Preliminary evidence from our laboratory suggested that over-expression of the cell cycle checkpoint gene, WEE1, repressed growth and development. Here the hypothesis is tested that the level of WEE1 has a dosage effect on growth and development in Arabidospis thaliana. To do this, a comparison was made of the development of gain- and loss-of-function WEE1 arabidopsis lines both in vivo and in vitro. METHODS: Hypocotyl explants from an over-expressing Arath;WEE1 line (WEE1(oe)), two T-DNA insertion lines (wee1-1 and wee1-4) and wild type (WT) were cultured on two-way combinations of kinetin and naphthyl acetic acid. Root growth and meristematic cell size were also examined. KEY RESULTS: Quantitative data indicated a repressive effect in WEE1(oe) and a significant increase in morphogenetic capacity in the two T-DNA insertion lines compared with WT. Compared with WT, WEE1(oe) seedlings exhibited a slower cell-doubling time in the root apical meristem and a shortened primary root, with fewer laterals, whereas there were no consistent differences in the insertion lines compared with WT. However, significantly fewer adventitious roots were recorded for WEE1(oe) and significantly more for the insertion mutant wee1-1. Compared with WT there was a significant increase in meristem cell size in WEE1(oe) for all three ground tissues but for wee1-1 only cortical cell size was reduced. CONCLUSIONS: There is a gene dosage effect of WEE1 on morphogenesis from hypocotyls both in vitro and in vivo.

Arabidopsis T-DNA insertional lines for CDC25 are hypersensitive to hydroxyurea but not to zeocin or salt stress.

BACKGROUND AND AIMS: In yeasts and animals, cyclin-dependent kinases are key regulators of cell cycle progression and are negatively and positively regulated by WEE1 kinase and CDC25 phosphatase, respectively. In higher plants a full-length orthologue of CDC25 has not been isolated but a shorter gene with homology only to the C-terminal catalytic domain is present. The Arabidopis thaliana;CDC25 can act as a phosphatase in vitro. Since in arabidopsis, WEE1 plays an important role in the DNA damage/DNA replication checkpoints, the role of Arath;CDC25 in conditions that induce these checkpoints or induce abiotic stress was tested. Methods arath;cdc25 T-DNA insertion lines, Arath;CDC25 over-expressing lines and wild type were challenged with hydroxyurea (HU) and zeocin, substances that stall DNA replication and damage DNA, respectively, together with an abiotic stressor, NaCl. A molecular and phenotypic assessment was made of all genotypes Key RESULTS: There was a null phenotypic response to perturbation of Arath;CDC25 expression under control conditions. However, compared with wild type, the arath;cdc25 T-DNA insertion lines were hypersensitive to HU, whereas the Arath;CDC25 over-expressing lines were relatively insensitive. In particular, the over-expressing lines consistently outgrew the T-DNA insertion lines and wild type when challenged with HU. All genotypes were equally sensitive to zeocin and NaCl. CONCLUSIONS: Arath;CDC25 plays a role in overcoming stress imposed by HU, an agent know to induce the DNA replication checkpoint in arabidopsis. However, it could not enhance tolerance to either a zeocin treatment, known to induce DNA damage, or salinity stress.

Ectopic expression of LEAFY COTYLEDON1-LIKE gene and localized auxin accumulation mark embryogenic competence in epiphyllous plants of Helianthus annuus x H. tuberosus.

BACKGROUND AND AIMS: The clone EMB-2 of the interspecific hybrid Helianthus annuus x H. tuberosus provides an interesting system to study molecular and physiological aspects of somatic embryogenesis. Namely, in addition to non-epiphyllous (NEP) leaves that expand normally, EMB-2 produces epiphyllous (EP) leaves bearing embryos on the adaxial surface. This clone was used to investigate if the ectopic expression of H. annuus LEAFY COTYLEDON1-LIKE (Ha-L1L) gene and auxin activity are correlated with the establishment of embryogenic competence. METHODS: Ha-L1L expression was evaluated by semi-quantitative RT-PCR and in situ hybridization. The endogenous level and spatial distribution of free indole-3-acetic acid (IAA) were estimated by a capillary gas chromatography-mass spectrometry-selected ion monitoring method and an immuno-cytochemical approach. KEY RESULTS: Ectopic expression of Ha-L1L was detected in specific cell domains of the adaxial epidermis of EP leaves prior to the development of ectopic embryos. Ha-L1L was expressed rapidly when NEP leaves were induced to regenerate somatic embryos by in vitro culture. Differences in auxin distribution pattern rather than in absolute level were observed between EP and A-2 leaves. More precisely, a strong IAA immuno-signal was detected in single cells or in small groups of cells along the epidermis of EP leaves and accompanied the early stages of embryo development. Changes in auxin level and distribution were observed in NEP leaves induced to regenerate by in vitro culture. Exogenous auxin treatments lightly influenced Ha-L1L transcript levels in spite of an enhancement of the regeneration frequency. CONCLUSIONS: In EP leaves, Ha-L1L activity marks the putative founder cells of ectopic embryos. Although the ectopic expression of Ha-L1L seems to be not directly mediated by auxin levels per se, it was demonstrated that localized Ha-L1L expression and IAA accumulation in leaf epidermis domains represent early events of somatic embryogenesis displayed by the epiphyllous EMB-2 clone.

Overexpression of KNAT1 in lettuce shifts leaf determinate growth to a shoot-like indeterminate growth associated with an accumulation of isopentenyl-type cytokinins.

Leaves are specialized organs characterized by defined developmental destiny and determinate growth. The overexpression of Knotted1-like homeobox genes in different species has been shown to alter leaf shape and development, but a definite role for this class of genes remains to be established. Transgenics that overexpress Knotted1-like genes present some traits that are characteristic of altered cytokinin physiology. Here we show that lettuce (Lactuca sativa) leaves that overexpress KNAT1, an Arabidopsis kn1-like gene, acquire characteristics of indeterminate growth typical of the shoot and that this cell fate change is associated with the accumulation of specific types of cytokinins. The possibility that the phenotypic effects of KNAT1 overexpression may arise primarily from the modulation of local ratios of different cytokinins is discussed.

Short- and long-term effects of dehydroascorbate in Lupinus albus and Allium cepa roots.

Administration of 1 mM dehydroascorbate (DHA) results in a rapid and large increase in cellular ascorbate (AA) content in both Lupinus albus L. and Allium cepa L. root tips. Uptake of DHA from the medium occurs at a high rate within 10-12 h of incubation, and is slowed down thereafter. In the first few h, DHA reduction to AA is apparently correlated to GSH depletion and slightly higher DHA reductase activity. DHA incubation also seems to induce new GSH synthesis. Longer DHA incubation (24 h) affects root growth by inhibiting cell proliferation. At this stage, an apparently generalised oxidation of SH-containing proteins is observed in DHA-treated roots. Treatment with 1 mM L-galactono-gamma-lactone, the last precursor of AA biosynthesis, results in an increase in AA content similar to that obtained with DHA, but stimulates growth and affects the redox state of SH-containing proteins in the opposite way. A possible multi-step mechanism of DHA reduction/removal is suggested and the hypothesis that DHA inhibits cell cycle progression by affecting the redox state of SH-containing proteins is discussed.

Inactivation of DNA replication origins by the cell cycle regulator, trigonelline, in root meristems of Lactuca sativa.

The effects of trigonelline (TRG) on the cell cycle in root meristems of Lactuca sativa L. were examined in the knowledge that TRG is a cell cycle regulator that causes cell arrest in G2, and prevents ligation of replicons in S-phase. The hypothesis was tested that continuous exposure to TRG would perturb DNA replication which, in turn, would lengthen the cell cycle and impair root elongation. Using DNA fibre autoradiography, mean replicon size was 31 and 13 microm in the TRG (3 mM) and control treatments, respectively. Trigonelline also resulted in a lengthening of both S-phase and the cell cycle and a decrease in primary root elongation. Hence, replicon inactivation was responsible for the protracted S-phase. Trigonelline treatment also resulted in a 1.6-fold increase in fork rate (13.8 microm h(-1)) compared with the control (8.4 m h(-1)). The faster fork rate in the larger replicons is in accord with the highly significant positive relationship already established between fork rate and replicon size for various unrelated higher plants.

Isolation and molecular characterisation of the gene encoding the cytoplasmic ribosomal protein S28 in Prunus persica [L.]] Batsch.

RT-PCR was performed on peach (Prunus persica [L.] Batsch) RNA to isolate cDNAs corresponding to transcripts which are differentially expressed in leaves borne on basal and apical shoots. A gene was identified which was more highly expressed in the leaves of basal shoots, and codes for the cytoplasmic protein S28 present in the small ribosomal subunit. The 5' leader regions of RPS28 mRNAs were found to harbour 8-11 pyrimidine tracts, which suggested similarities to regulatory stretches that control the translation of mRNAs for ribosomal proteins in animals. The peach S28 is encoded by two intron-containing genes, which are both transcribed in mitotically active tissues such as developing leaves and roots. In situ hybridisation to shoot vegetative apices and the measurement of nucleus/nucleolus ratios indicated that RPS28 expression was confined to areas undergoing active cell division. The mature RPS28 mRNA was detected as a single species in actively dividing tissues such as apical tips, developing leaves, vegetative buds, stamens, developing fruits and roots. In contrast, accumulation of a precursor RNA, in the presence of the mature product, was found in fully expanded leaves and subtending stems, while only the precursor species was detected in several late-stage tissues. This phenomenon suggested that expression of the mature RNA is controlled at the level of splicing and turnover of the precursor RNA. This is similar to the mode of regulation of ribosomal protein genes in animals.

Amount and organization of the heterochromatin in Olea europaea and related species.

The amount and spatial organization of the heterochromatin in nuclei of the shoot meristem and the frequency in the nuclear DNA of sequences belonging to a family of tandem repeats were investigated in cultivars of Olea europaea and related species. Significant differences between Olea species and between cultivars of O. europaea were observed: (i) in the spatial organization of the heterochromatin in interphase nuclei as determined by the number and surface area of the chromocentres; (ii) in genome size; and (iii) in the amount of condensed chromatin as measured by cytophotometry carried out at different thresholds of optical density. DNA elements belonging to a family of tandem repeats about 80 bp in length (OeTaq80 repeats) were isolated from the genomic DNA of an olive cultivar. It was shown: (i) by nucleotide sequence comparisons, that these repeats display variability in structure even within the same array, where different elements may share no more than 74% homology; (ii) by in situ hybridization, that OeTaq80-related DNA sequences are mainly localized in the heterochromatin at the chromosome ends; (iii) by dot-blot hybridization experiments, that these sequences are highly represented in the genome of all the olive cultivars and the majority of Olea species studied, and that their frequency may differ significantly even between olive cultivars; and (iv) by calculating the copy number of OeTaq80-related sequences per haploid (1C) genome, that the redundancy of these DNA elements may differ significantly between the genomes tested. It is suggested that the inter- and intraspecific changes in the nuclear and genomic traits observed can contribute to the understanding of the phylogenetic relationships between Olea species and in defining parameters to be exploited in varietal identification within cultivated olives.

Automatic computation of enzyme kinetics by HPLC.

Enzyme activity can be easily measured by HPLC using traces of the product itself as an internal standard. Our procedure involved the development of an equation using the experimental data obtained in the kinetic assay. The entire procedure can thus be automated and a computer program is presented here for facilitating the assay and saving time. The determination of the activity of NAD kinase is reported as an example.

The size of quiescent centre in roots of Allium cepa L. grown with ascorbic acid.

Following ascorbic-acid treatment a large number of quiescent centre cells undergo DNA synthesis and, at the same time, the cell proliferation in the entire root meristem of Allium cepa is stimulated. The effects of ascorbic acid on dividing cells in the meristem proper and on the quiescent centre are long-lasting since they are obtained in both short- and long-term experiments. Whatever the time of treatment with ascorbic acid and whatever the starting size of the quiescent centre (450 or 1000 cells), there is always a minimum number of quiescent centre cells (90-100) which remain in the G1 phase.

Relationship between ascorbic acid and cell division.

Proliferating cells require large amounts of ascorbic acid to reach cell division. The decrease in ascorbic acid caused by adding lycorine, an inhibitor of ascorbic acid biosynthesis, induces profound inhibition of cell division: the cell cycle is arrested in G1 and G2 phase, more than 90% of the cells being accumulated in G1 after some time. The effect of lycorine on mitotic index (MI) has been reversed by increasing experimentally the concentration of ascorbic acid in tissues. Ascorbic acid control on cell division is found to be specific, since isoascorbic acid is wholly ineffective. It is suggested that the principal role of ascorbic acid in the cell cycle may be related to its action in controlling the synthesis of hydroxyproline-containing proteins, which can be essential requirements for development of G1 and G2.