Search

Sugar beet leaves can be a viable and economically interesting source of high-quality protein for the food industry. We investigated how storage conditions and leaf wounding at harvest affect the content and quality of the soluble protein. After collection, leaves were either stored intact or shredded to mimic wounding induced by commercial leaf harvesters. Leaf material was stored in small volumes at different temperatures to assess leaf physiology or in larger volumes to assess temperature development at different locations in the bins. Protein degradation was more pronounced at higher storage temperatures. Wounding accelerated the degradation of soluble protein at all temperatures. Both wounding and storage at higher temperatures greatly stimulated respiration activity and heat production. At temperatures below 5°C, ribulose-1,5-biphosphate carboxylase oxygenase (RuBisCO) in intact leaves was preserved for up to 3 weeks. At temperatures of 30–40°C, RuBisCO degradation occurred within 48 h. Degradation was more pronounced in shredded leaves. In 0.8-m3 storage bins at ambient temperature, core temperatures rapidly increased, up to 25°C in intact leaves and up to 45°C in shredded leaves within 2–3 days. Immediate storage at 5°C greatly suppressed the temperature increase in intact but not in shredded leaves. The indirect effect of excessive wounding, that is, heat production, is discussed as the pivotal factor responsible for increased degradation of protein. For optimal retention of soluble protein levels and quality in harvested sugar beet leaves, it is advised to minimize wounding and to store the material at temperatures around −5°C. Practical Application: To preserve the soluble protein content and quality for at least 3 weeks, sugar beet leaves should be harvested with minimal wounding and stored at temperatures between 1 and 5°C. When aiming to store minimally wounded leaves in larger volumes, it must be ensured that the product temperature in the core of the biomass meets the temperature criterium or the cooling strategy must be adjusted. The principles of minimal wounding and low temperature storage are transferable to other leafy crops that are harvested for food protein.

The relations between physical and chemical characteristics (e.g., color, firmness, volatile and non-volatile metabolites) of red ripe strawberry fruit and the natural spoilage caused by Botrytis cinerea were investigated. The spoilage rates differed between genotypes, and this was highly correlated over two successive years. Among seventeen genotypes, a more intense red coloration of the fruit skin was associated with a lower spoilage rate (r = –0.63). Additionally, weakly negative correlations were found between the levels of anthocyanins, ascorbic acid, malic acid and spoilage rates. No clear correlations were found between spoilage rates and soluble sugars, most volatiles, firmness and dry weight percentage. High levels of two volatile compounds, ethyl butanoate (r = 0.55) and 1-hexanol (r = 0.61), were correlated to high spoilage rates. These characteristics may assist strawberry breeders in selecting for genotypes with reduced susceptibility to B. cinerea.

Since cucumbers suffer from a short postharvest life, applying different technologies is increasingly used as effective ways to increase their shelf life and quality. In this study a combination of chitosan-limonene coating and MAP storage has been used as a postharvest treatment to maintain cucumber quality. Samples were stored in three different packages: A (21% O2, macro-perforated package to be in equilibrium with air); package B (active MAP, starting concentrations 10% O2 + 5% CO2); package C (passive MAP starting concentrations 21% O2 + 0.1% CO2); they were stored at three temperatures (20, 10, and 4 °C). Quality parameters of cucumber such as weight loss, firmness, color, pH, fungal growth, Tg, organoleptic properties were determined. Interactive effects of coating, package, temperature, and storage time showed that coating and MAP in general had positive effects on several quality aspects. Coating combined with active MAP had the most positive effect on most postharvest attributes. However, using active MAP at higher temperature led to quality problems and is only useful if storage time is short. The combined usage of active MAP and chitosan-based coating on cucumber represents an innovative and interesting method for commercial application.

l -ascorbate (vitamin C, ASC) is an antioxidant that is essential for the proper function not only of plants but also animals. Light is a major regulatory factor for ASC levels in plants. In this paper, we review the regulation of ASC by light and the involved biochemical and physiological processes. Several biochemical pathways for ASC biosynthesis have been proposed to exist in plants. We aim to determine the contribution of these biochemical pathways on ASC levels and, locate the steps of them that are affected by light. From biochemical and genetic studies only evidence for ASC biosynthesis occurring via the d -mannose/L-galactose biosynthetic pathway was found. Alternative pathways might account for ASC biosynthesis only in transgenic plants. Apart from biosynthesis, recycling and turnover of ASC might affect the size of the ASC pool. Light regulation of ASC levels in plants occurs primarily via effects on biosynthesis. In addition, light affects ASC homeostasis and translocation within the plant. Light regulation of ASC has been studied for individual physiological processes without taking into account possible interactions. By establishing the physiological network behind light regulation of ASC for both leaves and fruit, we developed a novel hypothesis on interactions between the physiological processes that regulate ASC. We conclude that respiration and photosynthesis interact in light regulation of ASC biosynthesis via carbohydrate availability.

In this work, the involvement of programmed cell death (PCD) in the wound-induced postharvest browning disorder and senescence in butterhead lettuce (Lactuca sativa L.) fresh-cuts was studied. At the wounded (cut, bruised) sites, rapid browning, loss of chlorophyll and massive cell death, accompanied with accumulation of reactive oxygen species and increased electrolyte leakage occurred in a narrow strip of tissue adjacent the injury. The dead cell morphology (protoplast and nuclei shrinkage) together with the biochemical and physiological changes resembled necrotic PCD type. With a slight delay post-wounding, senescence associated with similar cell death features was initiated in distant non-wounded sites. In addition to necrotic PCD, both in wounded and senescing tissue, the appearance of empty cell corpses was observed, indicating that part of the cells might undergo vacuolar PCD (self-digestion of cellular content after vacuole collapse). The wounding-induced local cell death at the primary site of damage suggested that PCD may serve as a mechanism to seal-off the wound by building a physical barrier of dead cells. However, the cell death at sites remote from the wound suggests the distribution of long-distance senescence-inducing wound messengers. Trichomes in unwounded tissue often were the first to show H2O2 accumulation and dead cells; thereafter, the elevated H2O2 and cell death appeared in connecting cells and senescence progressed over larger areas. This suggests that trichomes may contribute to mediating the wound signalling leading to subsequent senescence. Our findings demonstrate that PCD is an integral part of the wound syndrome in fresh-cut lettuce.

Symptoms of water stress are the most frequent cause for the “end of vase life” in prior stored roses. It was hypothesized that dark storage may alter the stomatal functionality and may cause water balance problems during the subsequent vase life period. The effect of short- and long-term storage on functionality of stomatal and subsequent flower performance was investigated in two rose cultivars (cvs) (‘Akito’ and ‘Grand Prix’) with presumed different sensitivity for development of water stress symptoms during the vase life. Compared to no storage, both short term storage (2.3 d at 6 °C) and long term storage (28 d at 0.5 °C) negatively affected the stomatal functionality in cultivar (cv) Akito. Stomatal functionality parameters such as the rapidity of the closing response upon dehydration and the relative water content at which stomata are fully closed showed good correlations with flower performance parameters (flower weight changes and vase life). This indicates that in cv Akito, the decreased stomatal functionality is one of the factors involved in the poor vase life of prior stored flowers. In cv Grand Prix, however, storage did not greatly affect the stomatal functionality but storage negatively affected flower performance in a comparable way as in cv Akito. A pre-treatment with abscisic acid prior to storage slightly improved stomatal functionality in both cvs, but no clear effect on flower performance was observed. Addition of the bactericide 8-HQC to the vase water improved flower performance in both cvs but could not alleviate the negative effect of cold storage on flower performance. Results show that in roses cold storage may, depending on the cultivar, negatively affect stomatal functionality and this may contribute to water stress and ultimately flower failure. In addition, cold storage may negatively affect xylem water conducting properties through processes not related to bacterial contamination.

This study deals with quantifying sugar and acids levels important for the perceived taste of tomatoes (Solanum lycopersicum). Sugar and acids levels were measured repeatedly on the same tomato using tissue samples obtained with a biopsy needle in combination with HPLC protocols. Biopsies of pericarp and locular gel tissue from tomatoes differing in position in the truss, from mature green to ripe red, were taken from a beef- (‘Licorossa’), a cocktail- (‘Lucino’) and a cherry type (‘Petit Sweet’) cultivar. Tomatoes were stored up to three weeks at three temperatures (12, 19 and 24.5 °C) and biopsy samples were taken every few days. A model regarding the most important processes that interconvert sugars and acids (glycolysis, TCA cycle and gluconeogenesis (GNG)) is proposed. Results of the model calibration showed more breakdown of hexoses in red tomatoes and more conversion of malate into hexoses in green tomatoes. More hexose turnover was found in locular gel than in pericarp tissue. GNG was more important in the cherry type cultivar due to faster hexose and malate breakdown. In the round type cultivar malate levels were higher due to faster citrate breakdown and slower malate breakdown. Starch and sucrose levels did not significantly affect postharvest sugar and acid development. Molecular markers that quantify the kinetic parameters of the model might be important to develop genotypes with better taste performance.

The availability of rapid and accurate methods to assess fruit flavor is of utmost importance to support quality control especially in the breeding phase. Breeders need more information and analytical tools to facilitate selection for complex multigenic traits such as flavor quality. In this study, it is shown that proton-transfer reaction mass spectrometry (PTR-MS) is a suitable method to monitor at high sensitivity the emission of volatiles determining the tomato aromatic profile such as hexanal, hexenals, methanol, ethanol, and acetaldehyde. The volatiles emitted by 14 tomato varieties (at red stage) were analyzed by 2 solvent-free headspace methods: solid-phase microextraction/gas chromatography MS and PTR-MS. Multivariate statistics (principal component analysis and cluster analysis) of the PTR-MS results allow an unambiguous separation between varieties, especially with a clear fingerprinting separation between the different tomato types: round truss, cocktail, and cherry tomatoes. PTR-MS was also successfully used to monitor the changes in volatile profiles during postharvest ripening and storage.

Fruit and vegetables are regularly stored by consumers in the refrigerator at temperatures that may be well below the recommended storage temperatures. Apart from causing visible symptoms such as watery, sunken areas on the skin, chilling may also induce changes in fruit textural properties and flavour. The aim of this research was to investigate the effect of low temperature storage on tomato flavour and off-flavour production. To more closely mimic the real-consumer aroma perception while eating, in addition to the standard solid-phase microextraction gas chromatography-mass spectrometry (SPME/GC-MS) analysis, volatiles were also measured using a chewing device connected to a proton-transfer reaction-mass spectrometer (PTR-MS). Aroma volatiles were assessed in red ripe tomatoes of the cvs Cappricia RZ (round truss) and Amoroso RZ (cocktail truss) stored at refrigerator temperature (4 °C) and at higher temperatures (16 and 22 °C) for 20 days. The changes in aroma production were also monitored when the fruit was brought from room to refrigerator temperature and vice versa. After bringing the fruit from room to refrigerator temperature, the abundance of most volatiles was greatly reduced within 3 to 5 h, closely following the decrease in fruit temperature. When temperature was restored to room temperature following varying times of cold storage, the abundance of most volatiles increased again, but generally not to the original levels. Overall, the effects of low temperature storage on the decrease in volatile abundance were more pronounced in cv Cappricia RZ than in cv Amoroso RZ. On the contrary, the production of off flavours following prolonged cold storage was more pronounced in cv Amoroso RZ than in cv Cappricia RZ. Apart from changes in the overall abundance of the volatiles, marked changes in the volatile profile were observed in fruit stored for longer times in the cold and this may at least in part explain the negative effect of cold storage on overall tomato flavour.

In this study, important quality properties such as firmness, sugar and acid levels were measured and analysed in tomatoes harvested from three greenhouses during a five month period and stored at 16 degrees C for over 20 days. Tomatoes were harvested from three identical, neighbouring, greenhouses which were either conventionally ventilated (open greenhouse) or mechanically cooled (semi-closed greenhouses). Sugar and acids levels were hardly affected by greenhouse type. Compared to the open greenhouse, semi-closed greenhouses produced heavier and less mature (firmer) fruit at the commercial harvesting stage based on colour. Fruit maturity differences could be linked to the vertical temperature gradient and to CO2 levels in the different greenhouses. This indicates that CO2 levels and temperature affect the synchronisation between colour and firmness maturity at harvest. The acceptance period, i.e., the time period both tomato colour and firmness are considered acceptable by consumers, will likely be positively affected when growers switch from conventionally ventilated to semi-closed production systems. Additional to greenhouse effects also effects of the harvest month were observed. The sugar to acid ratio was highest and glucose to fructose ratio was lowest in July, the month with the highest irradiance, irrespective of greenhouse type. The estimated value for the maximum firmness (F-max) varied from 17.9 N in August to 31.2 N in June. This monthly variation in F-max explains an important part of the variation found in the postharvest behaviour of tomatoes. Interestingly, the monthly variation in F-max showed the same trend as found for the monthly initial sugar levels. It might be hypothesised that the monthly variation in glucose and fructose levels causes variation in that part of firmness that is generated by cell turgor. The monthly variation in F-max, sugar and acid levels could not be linked to climate conditions and remains to be elucidated. (C) 2013 Elsevier B.V. All rights reserved.

Phospholipase D (PLD) and its product phosphatidic acid (PA) are incorporated in a complex metabolic network in which the individual PLD isoforms are suggested to regulate specific developmental and stress responses, including plant programmed cell death (PCD). Despite the accumulating knowledge, the mechanisms through which PLD/PA operate during PCD are still poorly understood. In this work, the role of PLD alpha 1 in PCD and the associated caspase-like proteolysis, ethylene and hydrogen peroxide (H2O2) synthesis in tomato suspension cells was studied. Wild-type (WT) and PLD alpha 1-silenced cell lines were exposed to the cell death-inducing chemicals camptothecin (CPT), fumonisin B1 (FB1) and CdSO4. A range of caspase inhibitors effectively suppressed CPT-induced PCD in WT cells, but failed to alleviate cell death in PLD alpha 1-deficient cells. Compared to WT, in CPT-treated PLD alpha 1 mutant cells, reduced cell death and decreased production of H2O2 were observed. Application of ethylene significantly enhanced CPT-induced cell death both in WT and PLD alpha 1 mutants. Treatments with the PA derivative lyso-phosphatidic acid and mastoparan (agonist of PLD/PLC signalling downstream of G proteins) caused severe cell death. Inhibitors, specific to PLD and PLC, remarkably decreased the chemical-induced cell death. Taken together with our previous findings, the results suggest that PLD alpha 1 contributes to caspase-like-dependent cell death possibly communicated through PA, reactive oxygen species and ethylene. The dead cells expressed morphological features of PCD such as protoplast shrinkage and nucleus compaction. The presented findings reveal novel elements of PLD/PA-mediated cell death response and suggest that PLD alpha 1 is an important factor in chemical-induced PCD signal transduction.

Tomatoes are mostly harvested at the orange and red-ripe stages. A survey among consumers indicated that tomatoes are most often stored in the refrigerator well below 10 °C, a temperature considered harmful for chilling sensitive products such as tomato. Also during distribution, tomatoes may be exposed to chilling temperatures. The effects of storage at chilling temperatures on quality aspects of tomatoes were investigated. The colour and lycopene content of red ripe tomatoes of two cultivars (cocktail and round type) was evaluated during 20 days of storage at 4, 8, 12 and 16 °C. Colour was repeatedly measured over time by tristimulus colour measurements, RGB image analysis and colour intensity was scored by eye using a consumer panel. Lycopene content was repeatedly assessed by following the NAI index over time. This index, obtained from remittance VIS spectroscopy, was found to relate closely to the lycopene level as measured by HPLC measurements of pericarp tissue. Temperatures below 12 °C resulted in lycopene loss in ripe-red tomatoes and substantial colour loss well assessed by visual evaluation. Colour measurement using tristimulus colour measurements and RGB image analysis did not correlate well with lycopene content. Prior hot water treatment did not prevent lycopene loss. Results show that storage of red ripe tomatoes at chilling temperatures reduces the nutritional and presumed health promoting value and affects fruit visual quality.

Programmed cell death (PCD) is an integral part of plant development and of responses to abiotic stress or pathogens. Although the morphology of plant PCD is, in some cases, well characterised and molecular mechanisms controlling plant PCD are beginning to emerge, there is still confusion about the classification of PCD in plants. Here we suggest a classification based on morphological criteria. According to this classification, the use of the term 'apoptosis' is not justified in plants, but at least two classes of PCD can be distinguished: vacuolar cell death and necrosis. During vacuolar cell death, the cell contents are removed by a combination of autophagy-like process and release of hydrolases from collapsed lytic vacuoles. Necrosis is characterised by early rupture of the plasma membrane, shrinkage of the protoplast and absence of vacuolar cell death features. Vacuolar cell death is common during tissue and organ formation and elimination, whereas necrosis is typically found under abiotic stress. Some examples of plant PCD cannot be ascribed to either major class and are therefore classified as separate modalities. These are PCD associated with the hypersensitive response to biotrophic pathogens, which can express features of both necrosis and vacuolar cell death, PCD in starchy cereal endosperm and during self-incompatibility. The present classification is not static, but will be subject to further revision, especially when specific biochemical pathways are better defined.

Sliced tomatoes rapidly become translucent and show accelerated softening. Slicing-induced development of translucency and firmness decay of the outer pericarp was investigated in slices with or without locular gel and in slices from different tomato genotypes varying in their amount of locular gel. In addition, the effect of wounding-associated juice release on translucency and softening was investigated in slices stored as “single slice” and stored as “stacked slices” and in slices blotted dry immediately after wounding. Rapid translucency development was severely affected by the presence of locular gel. In slices where the locular gel was removed, and in cultivars with low amounts of locular gel, translucency development was much less. Translucency development seemed unrelated to the released juice from wounded tissue as it was not affected by the storage method (stacked or single slices) nor by dry blotting of cut surfaces immediately after slicing. Rapid firmness decay in slices is not related to the presence of the locular gel and was also not affected by storage method (stacked or single slices) nor by dry blotting of cut surfaces immediately after slicing The results show that translucency development and firmness decay are independent processes occurring simultaneously after slicing.

Botrytis cinerea is a fungal pathogen that greatly reduces the postharvest quality of rose flowers. A posthar- vest dip in 200 LL −1 sodium hypochlorite (NaOCl) for 10 s at 20 ◦C provided the greatest control of B. cinerea on 'Akito' and 'Gold Strike' flowers. NaOCl derived from Clorox ® Ultra household bleach solu- tion was more effective than laboratory grade NaOCl in reducing disease symptoms. Lowering the pH of the NaOCl solution from pH 9.7 (unadjusted) to pH 7.0 greatly improved its efficacy. Treating 'Gold Strike' flowers in this pH-adjusted NaOCl solution was more effective in reducing the level of infection on petals than postharvest dips in the conventional fungicides Medallion®, Phyton®, Switch® and Vangard®. Applying NaOCl prior to a 3- or 10-d commercial shipment also provided the most consistent disease control for a wide range of rose cultivars as compared to conventional fungicides. Of particular interest, the efficacy of NaOCl and Phyton ® was greatest when these compounds were applied to 'Gold Strike' flowers after incubation at 20 ◦C and 90% RH for 6-9 h. Our findings highlight NaOCl as a promising new candidate for the control of B. cinerea on rose flowers.

Detached tobacco leaves were infiltrated with an AT toxin preparation from the foliar pathogen Alternaria alternata tobacco pathotype. The AT toxin preparation caused formation of necrotic lesions within 5 days post-infiltration in a concentration-dependent manner. Cell death was accompanied by increased levels of the stress metabolites hydrogen peroxide, malondialdehyde, free proline and by enhanced total protease activity. Lesion development and the production of stress metabolites were suppressed if the infiltration site was pre-infiltrated with caspase-specific peptide inhibitors (irreversible caspase-1 inhibitor acyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-CMK) and the broad range caspase inhibitor benzyoxycarbonyl-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp-CH2-DCB)), the serine protease inhibitor N alpha-p-tosyl-L-lysine chloromethylketone and the polyamine spermine. Extensive accumulation of reactive oxygen species (ROS), as determined by staining with 3-3'-diaminobenzidine and 2',7'-dichlorofluorescein diacetate, was found in the AT toxin-challenged lesions. The data show that AT toxin-induced cell death in tobacco is a type of programmed cell death in which caspase-like proteases and ROS signalling play a prominent role.

SUMMARY Susceptibility was evaluated of host and non-host plants to three pathogenic Botrytis species: the generalist B. cinerea and the specialists B. elliptica (lily) and B. tulipae (tulip). B. tulipae was, unexpectedly, able to infect plant species other than tulip, and to a similar extent as B. cinerea. To study host and non-host interactions in more detail, the three Botrytis species were inoculated on Arabidopsis wild-types and 23 mutant genotypes. Disease development was monitored macroscopically by quantifying the lesion area and microscopically by bright-field and fluorescence microscopy following histochemical staining. B. cinerea and B. tulipae were very similar in their ability to infect the tested Arabidopsis genotypes, whereas B. elliptica caused disease only on a few Arabidopsis mutant genotypes. Arabidopsis mutants with a delayed or reduced cell death response were generally more resistant to Botrytis infection, whereas mutants in which cell death was accelerated were more susceptible. Differences in susceptibility between genotypes were generally gradual. Only the camalexin-deficient mutant pad3 was fully susceptible to all three Botrytis species. Cellular changes were monitored during compatible and incompatible interactions. The formation of papillae, the presence of lysosome-like vesicles and the intracellular accumulation of H(2)O(2) and nitric oxide were visualized in the infection zones using fluorescent probes. Based on histology and responses of Arabidopsis mutants, a model is proposed in which resistance against Botrytis, besides the production of camalexin, depends on the balance between cell death and survival.

For a vast number of ornamental species, blocking the plant's response to ethylene is an efficient strategy to enhance the longevity of the flowers. The most effective ways to conduct such interference will be reviewed in this paper. A large number of chemical compounds have been evaluated for their effects on ethylene production and perception. Among these are a range of strained olefines. This has resulted in the discovery that cyclopropenes, among them 1-methylcyclopropene (1-MCP) and a number of other substituted cyclopropenes effectively block ethylene responses at the receptor level. A lot of testing remains to be done to uncover the full potential of these compounds, but they do offer promising new ways to extend the postharvest life of ornamentals. Also genetic modification appears to be a very effective way in controlling of ethylene synthesis and perception. Attempts to use both a reduced endogenous ethylene production and a reduced sensitivity to ethylene will be reviewed. Among these the use of the mutant ethylene receptor gene, etr1-1, from Arabidopsis seems most promising, especially when it is expressed under the control of a flower specific promoter.

Flower senescence is accompanied by the rapid death of large numbers of cells. In situ DNA degradation was studied in gypsophila (Gypsophila paniculata) petals during flower opening and senescence, using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). In flowers that had just opened, TUNEL staining was exclusively observed in nuclei in the vicinity of (developing) vessels, presumably reflecting xylem vessel differentiation. In fully open flowers, TUNEL positive nuclei appear in all the cells well before the increase in ethylene production and visible signs of senescence. Occurrence of TUNEL positive nuclei was stimulated by ethylene but not prevented by silver thiosulphate. The results show that gypsophila petal senescence is a form of PCD with features of animal apoptosis and suggest that nuclear DNA degradation is an early regulatory event rather than a result of massive cell death in the final stage of senescence.

Programmed cell death (PCD) is an integral part of plant development and defence. It occurs at all stages of the life cycle, from fertilization of the ovule to death of the whole plant. Without it, tall trees would probably not be possible and plants would more easily succumb to invading microorganisms. Here, we have attempted to categorize plant PCD in relation to three established morphological types of metazoan cell death: apoptosis, autophagy and non-lysosomal PCD. We conclude that (i) no examples of plant PCD conform to the apoptotic type, (ii) many examples of PCD during plant development agree with the autophagic type, and (iii) that other examples are apparently neither apoptotic nor autophagic.

The regulation of gravistimulation-induced ethylene production and its role in gravitropic bending was studied in Antirrhinum majus L. cut flower stems. Gravistimulation increased ethylene production in both lower and upper halves of the stems with much higher levels observed in the lower half. Expression patterns of three different 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) genes, an ACC oxidase (ACO) and an ethylene receptor (ETR/ERS homolog) gene were studied in the bending zone of gravistimulated stems and in excised stem sections following treatment with different chemicals. One of the ACS genes (Am-ACS3) was abundantly expressed in the bending zone cortex at the lower side of the stems within 2 h of gravistimulation. Am-ACS3 was not expressed in vertical stems or in other parts of (gravistimulated) stems, leaves or flowers. Am-ACS3 was strongly induced by indole-3-acetic acid (IAA) but not responsive to ethylene. The Am-ACS3 expression pattern strongly suggests that Am-ACS3 is responsible for the observed differential ethylene production in gravistimulated stems; its responsiveness to IAA suggests that Am-ACS3 expression reflects changes in auxin signalling. Am-ACS1 also showed increased expression in gravistimulated and IAA-treated stems although to a much lesser extent than Am-ACS3. In contrast to Am-ACS3, Am-ACS1 was also expressed in non-bending regions of vertical and gravistimulated stems and in leaves, and Am-ACS1 expression was not confined to the lower side cortex but evenly distributed over the diameter of the stem. Am-ACO and Am-ETR/ERS expression was increased in both the lower and upper halves of gravistimulated stems. Expression of both Am-ACO and Am-ETR/ERS was responsive to ethylene, suggesting regulation by IAA-dependent differential ethylene production. Am-ACO expression and in vivo ACO activity, in addition, were induced by IAA, independent of the IAA-induced ethylene. IAA-induced growth of vertical stem sections and bending of gravistimulated flowering stems were little affected by ethylene or 1-methylcyclopropene treatments, indicating that the differential ethylene production plays no pivotal role in the kinetics of gravitropic bending.

Programmed cell death (PCD) in plant cells is often accompanied by biochemical and morphological hallmarks similar to those of animal apoptosis. However, orthologs of animal caspases, cysteinyl aspartate-specific proteases that constitute the core component of animal apoptosis, have not yet been identified in plants. Recent studies have revealed the presence of a family of genes encoding proteins with distant homology to mammalian caspases, designated metacaspases, in the Arabidopsis thaliana genome. Here, we describe the isolation of LeMCA1, a type-II metacaspase cDNA clone from tomato (Lycopersicon esculentum Mill.). BLAST analysis demonstrated that the LeMCA1 gene is located in close vicinity of several genes that have been linked with PCD. Southern analysis indicated the existence of at least one more metacaspase in the tomato genome. LeMCA1 mRNA levels rapidly increased upon infection of tomato leaves with Botrytis cinerea, a fungal pathogen that induces cell death in several plant species. LeMCA1 was not upregulated during chemical-induced PCD in suspension-cultured tomato cells.

Camptothecin, a topo isomerase-I inhibitor used in cancer therapy, induces apoptosis in animal cells. In tomato (Lycopersicon esculentum Mill.) suspension cells, camptothecin induces cell death that is accompanied by the characteristic nuclear morphological changes such as chromatin condensation and nuclear and DNA fragmentation that are commonly associated with apoptosis in animal systems. These effects of camptothecin can effectively be blocked by inhibitors of animal caspases, indicating that, in tomato suspension cells, camptothecin induces a form of programmed cell death (PCD) with similarities to animal apoptosis (A.J. De Jong et al. (2000) Planta 211:656-662). Camptothecin-induced cell death was employed to study processes involved in plant PCD. Camptothecin induced a transient increase in H2O2 production starting within 2 h of application. Both camptothecin-induced cell death and the release of H2O2 were effectively blocked by application of the calcium-channel blocker lanthanum chloride, the caspase-specific inhibitor Z-Asp-CH2-DCB, or the NADPH oxidase inhibitor diphenyl iodonium, indicating that camptothecin exerts its effect on cell death through a calcium- and caspase-dependent stimulation of NADPH oxidase activity. In addition, we show that ethylene is an essential factor in camptothecin-induced PCD. Inhibition of either ethylene synthesis or ethylene perception by L-alpha-(2-aminoethoxyvinyl)glycine or silver thiosulphate, respectively, blocked camptothecin-induced H2O2 production and PCD. Although, in itself, insufficient to trigger H2O2 production and cell death, exogenous ethylene greatly stimulated camptothecin-induced H2O2 production and cell death. These results show that ethylene is a potentiator of the camptothecin-induced oxidative burst and subsequent PCD in tomato cells. The possible mechanisms by which ethylene stimulates cell death are discussed.

A new system to study programmed cell death in plants is described. Tomato (Lycopersicon esculentum Mill.) suspension cells were induced to undergo programmed cell death by treatment with known inducers of apoptosis in mammalian cells. This chemical-induced cell death was accompanied by the characteristic features of apoptosis in animal cells, such as typical changes in nuclear morphology, the fragmentation of the nucleus and DNA fragmentation. In search of processes involved in plant apoptotic cell death, specific enzyme inhibitors were tested for cell-death-inhibiting activity. Our results showed that proteolysis plays a crucial role in apoptosis in plants. Furthermore, caspase-specific peptide inhibitors were found to be potent inhibitors of the chemical-induced cell death in tomato cells, indicating that, as in animal systems, caspase-like proteases are involved in the apoptotic cell death pathway in plants.

In Petunia hybrida flowers, both pollination and stigma wounding induced a transient increase in ethylene pro­ duction and hastened corolla senescence. Ethylene production by different flower parts was measured in situ using laser photoacoustic (LPA) spectroscopy. In pollinated flowers, ethylene was exclusively produced by the stigma/style region whereas wounding of the stigma induced ethylene production both by the stigma/style region and by the remaining flower parts. In aminoethoxyvinylglycine (AVG)-treated flowers, sub­ sequent treatment of the unwounded stigma with 1«aminocyclopropane-1-carboxylic acid (ACC) induced ethylene production exclusively by the stigma/style region whereas treatment of a previously wounded stigma with ACC induced a simultaneous increase in ethylene production by the stigma/style region and the remaining flower parts. These results suggest that following stigma wounding, either ACC or ethylene is involved in inter-organ communication. Following pol­ lination, the signal is apparently not directly related to ethylene.

Ethylene production and expression patterns of an 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (CARAO1) and of two ACC synthase (EC 4.4.1.14) genes (CARACC3 and CARAS1) were studied in floral organs of cut carnation flowers (Dianthus caryophyllus L.) cv. White Sim. During the vase life and after treatment of fresh flowers with ethylene, production of ethylene and expression of ethylene biosynthetic genes first started in the ovary followed by the styles and the petals. ACC oxidase was expressed in all the floral organs whereas, during the vase life, tissue-specific expression of the two ACC synthase genes was observed. After treatment with a high ethylene concentration, tissue specificity of the two ACC synthase genes was lost and only a temporal difference in expression remained. In styles, poor correlation between ethylene production and ACC synthase (CARAS1) gene expression was observed suggesting that either activity is regulated at the translational level or that the CARAS1 gene product requires an additional factor for activity. Isolated petals showed no increase in ethylene production and expression of ethylene biosynthetic genes when excised from the flower before the increase in petal ethylene production (before day 7); showed rapid cessation of ethylene production and gene expression when excised during the early phase of petal ethylene production (day 7) and showed a pattern of ethylene production and gene expression similar to the pattern observed in the attached petals when isolated at day 8. The interorgan regulation of gene expression and ethylene as a signal molecule in flower senescence are discussed.

Ozone-induced changes in ethylene production, ACC oxidase activity and the contents of ACC, MACC and free PAs were studied inPopulus nigra L. cv. Loenen with high ozone sensitivity as judged by the degree of chlorophyll degradation and premature leaf abscission.Ethylene production, ACC oxidase activity, ACC content and MACC levels were induced by the one-, two-, and three-week ozone exposure (36±9 ppb O3 for 11 hours a day). In addition, increases in PA levels, especially in spermidine, were measured in ozone treated plants.The role of free PAs and MACC synthesis as possible antisenescence reactions is discussed.

Le mecanisme de regulation de la senescence des petales est examine dans la fleur de Cymbidium. Lors de l'induction d'une senescence precoce par emasculation, les variations dans la production d'ethylene par la fleur sont observees ainsi que la concentration interne en ethylene, le contenu en acide 1-aminocyclopropane-1-carboxylique (ACC), en malonyl-ACC et l'activite de l'enzyme productrice d'ethylene dans les differentes parties de la fleur

Degenerate oligonucleotides to highly conserved regions of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14), the key enzyme in ethylene biosynthesis, were used to prime the synthesis and amplification of fragments of about 1,180 bp by polymerase chain reaction (PCR) in samples of cDNA to total RNA isolated from senescing carnation (Dianthus caryophyllus) flowers. Two putative ACC synthase PCR clones were isolated one of which was identical to the sequence of a carnation ACC synthase cDNA clone (CARACC3) recently isolated by Park et al. (Plant Mol Biol 18 (1992) 377-386). The other clone (CARAS1) was ca. 66% homologous at the amino acid level to CARACC3. For both ACC synthase clones, specific oligonucleotides were synthesized and, using PCR, we were able to distinguish between the two ACC synthase transcripts in samples of total RNA isolated from different carnation flower parts and leaves. DNA blots of PCR fragments revealed that, in flowers, both ageing and ethylene stimulated the occurrence of these transcripts in an organ-specific way. CARACC3 was more abundant in RNA from the petals whereas CARAS1 was more abundant in RNA from the styles. Despite a high ethylene production observed in ovaries, the level of both transcripts was low, suggesting the existence of a third ACC synthase gene that is specifically expressed in the ovary. Transcript levels in leaves were low irrespective of treatment.

The effects of relative humidity (RH) and of treatment of the rostellum with aminoethoxyvinylglycine (AVG), 1-aminocyclopropane-l-carboxylic acid (ACC) and water-insoluble vacuum grease on emasculation-induced phenomena in Cymbidium and Phalaenopsis flowers were investigated. Under conditions of high RH and after treatment of the rostellum with water-insoluble grease, the normal response to emasculation (i.e. increased ethylene production, lip coloration and wilting) was absent. However, under conditions of high RH this response could be restored by the addition of 2 0 nmol ACC on to the rostellar surface. Under conditions of low RH the response was inhibited by AVG; this inhibition was partially reversed by addition of 2 0 nmol ACC. The data provide evidence that desiccation of the rostellum is responsible for post-emasculation phenomena in orchid flowers.

Woltering, E. J. and Van Doom, W. G. 1988. Role of ethylene in senescence of petals—morphological and taxonomical relationships.—J. exp. Bot. 39: 1605-1616. Petal senescence in mature flowers was studied in 93 species from 22 families. The initial symptom of senescence was either wilting or abscission, but in some species the time span between wilting and abscission was very short. There was no apparent relationship between corolla form (choripetalous or sympetalous), ovary position (inferior or superior with respect to the corolla) and type of senescence (initial wilting or initial abscission). In monocots no initial abscission was found, while in dicots the difference between the wilting type and the abscission type was generally at the family level. With respect to petal senescence, sensitivity to exogenous ethylene (C2H4) was also related to the family level. Except for a few families (all tested Campanulaceae, Caryophyllaceae and Malvaceae, and most Orchidaceae), most of the flowers investigated that showed initial wilting were not sensitive to exogenous ethylene, e.g. all tested Compositae, Iridaceae, and Liliaceae. Most of the flowers showing initial abscission were sensitive to exogenous ethylene (Geraniaceae, Labiatae, Ranunculaceae, Rosaceae, Scrophulariaceae). Experiments with silver thiosulphate (STS) confirmed the effects of exogenous ethylene, both in flowers showing initial wilting and in flowers showing initial abscission. The data indicate, therefore, that ethylene is involved in the natural senescence of only a minority of the wilting type of flowers and in a majority (if not all) of the abscising type of flowers. Key words—Abscission, ethylene, senescence, silver thiosulphate.