Wouter G. van Doorn

Physiology and molecular biology of petal senescence

Petal senescence is reviewed, with the main emphasis on gene expression in relation to physiological functions. Autophagy seems to be the major mechanism for large-scale degradation of macromolecules, but it is still unclear if it contributes to cell death. Depending on the species, petal senescence is controlled by ethylene or is independent of this hormone. EIN3-like (EIL) transcription factors are crucial in ethylene-regulated senescence. The presence of adequate sugar levels in the cell delays senescence and prevents an increase in the levels of EIL mRNA and the subsequent up-regulation of numerous senescence-associated genes. A range of other transcription factors and regulators are differentially expressed in ethylene-sensitive and ethylene-insensitive petal senescence. Ethylene-independent senescence is often delayed by cytokinins, but it is still unknown whether these are natural regulators. A role for caspase-like enzymes or metacaspases has as yet not been established in petal senescence, and a role for proteins released by organelles such as the mitochondrion has not been shown. The synthesis of sugars, amino acids, and fatty acids, and the degradation of nucleic acids, proteins, lipids, fatty acids, and cell wall components are discussed. It is claimed that there is not enough experimental support for the widely held view that a gradual increase in cell leakiness, resulting from gradual plasma membrane degradation, is an important event in petal senescence. Rather, rupture of the vacuolar membrane and subsequent rapid, complete degradation of the plasma membrane seems to occur. This review recommends that more detailed analysis be carried out at the level of cells and organelles rather than at that of whole petals.

Relationship between browning and the activities of polyphenol oxidase and phenylalanine ammonia lyase in banana peel during low temperature storage

Kluai Khai (Musa AA Group) and Kluai Hom Thong (Musa AAA Group) bananas were stored at 6 and 10 °C. Visible chilling injury (CI) in the peel, mainly browning, occurred at both temperatures, but more so at 6 °C, and without significant differences between the cultivars. At the time of harvest, total free phenolics in the peel were three times lower in Kluai Khai than in Kluai Hom Thong fruit, the polyphenol oxidase (PPO) activity in Kluai Khai being considerably higher and phenylalanine ammonia lyase (PAL) activity much lower. As CI developed, PAL and PPO activities in the peel increased, and total free phenolics decreased. The decrease in total free phenolic compounds and the increase in PAL and PPO activities occurred more rapidly at 6 °C than at 10 °C, in both banana cultivars. Correlations between visible CI and the level of total free phenolics, and between CI and the activities of PPO and PAL, were all highly significant. The results indicate that low temperature stress induced concerted activities of PAL and PPO, which resulted in browning. Since the concentrations of free phenolic compounds and the rate of PAL and PPO activities varied considerably between the two cultivars, but browning did not, the changes in the biochemical parameters rather than their absolute levels were correlated with peel browning.

Role of endogenous bacteria in vascular blockage of cut rose flowers

Summary When cut flowers of five rose varieties (Sonia, Ilona, Motrea, Jack Frost and Mercedes) were held in water for 7 days, lowest hydraulic conductance was found in the basal 5 cm stem segment. After artificially blocking about two thirds of the xylem vessels in cut Sonia roses, the uptake of water by the stem was not reduced. This indicates that vascular occlusion involves a majority of the xylem vessels. Hydraulic conductance was related to the number of endogenous bacteria but was only significantly lowered when the number of bacteria in the 5 cm segments exceeded 106 cfu per gram fresh weight. Bacteriostatic chemicals (AgN03, benzalkone, HQC, DICA) inhibited growth of bacteria in stems and prevented the decrease of hydraulic conductance. The effect of AgN03 (also an anti-ethylene agent) on hydraulic conductance was related to the number of endogenous bacteria. HQC inhibited production of ethylene by the cut surface of rose stems, but had the same effect on hydraulic conductance as DICA and benzalkone, which stimulated ethylene production with respect to controls. The data indicate that vascular blockage was not due to ethylene production by the cut surface nor due to other physiological processes occurring after cutting of the xylem tissue. Instead, the data suggest that vascular blockage was mainly due to the presence of bacteria.

Is the onset of senescence in leaf cells of intact plants due to low or high sugar levels

This review examines the hypotheses that developmental programmed cell death in leaves is mediated (i) by sugar starvation in the leaf cells or (ii) by sugar accumulation in these cells. Experimental evidence for both hypotheses is critically discussed and found to be lacking. For example, some papers show that sugars prevent senescence of cut leaves placed in darkness, and prevent low sugar levels in the leaves. In these tests, the sugars seem to replace photosynthesis, hence the results have little relevance to leaf senescence in intact plants in the light. Low nitrogen nutrition and high light results in earlier senescence than the low nitrogen treatment alone. This is accompanied by high sugar levels in the leaves. The results have led to the idea that accumulation of sugars is the cause of the additional effect, or more generally, that sugar accumulation is always the direct cause of leaf senescence. Results from over-expressing, or knocking out, hexokinase genes tend to support the high sugar hypothesis, but pleiotropic effects confound this conclusion. In addition, several experiments show the effects of treatments on senescence without the increase in leaf sugar levels. Nonetheless, sugar levels are usually measured in whole leaves. Such an overall level does not reflect the differences in the onset of senescence between tissues and cells, and can therefore not be used as an argument for or against either of the two hypotheses. It is argued that future work should determine the time line of the concentrations of various sugars in various cells and cellular compartments, in relation to senescence processes in the same cells. Taken together, the data are not decisive. It is possible that neither of the two hypotheses is correct.

Nuclear fragmentation and DNA degradation during programmed cell death in petals of morning glory (Ipomoea nil)

We studied DNA degradation and nuclear fragmentation during programmed cell death (PCD) in petals of Ipomoeanil (L.) Roth flowers. The DNA degradation, as observed on agarose gels, showed a large increase. Using DAPI, which stains DNA, and flow cytometry for DAPI fluorescence, we found that the number of DNA masses per petal at least doubled. This indicated chromatin fragmentation, either inside or outside the nucleus. Staining with the cationic lipophilic fluoroprobe DiOC6 indicated that each DNA mass had an external membrane. Fluorescence microscopy of the nuclei and DNA masses revealed an initial decrease in diameter together with chromatin condensation. The diameters of these condensed nuclei were about 70% of original. Two populations of nuclear diameter, one with an average diameter about half of the other, were observed at initial stages of nuclear fragmentation. The diameter of the DNA masses then gradually decreased further. The smallest observed DNA masses had a diameter less than 10% of that of the original nucleus. Cycloheximide treatment arrested the cytometrically determined changes in DNA fluorescence, indicating protein synthesis requirement. Ethylene inhibitors (AVG and 1-MCP) had no effect on the cytometrically determined DNA changes, suggesting that these processes are not controlled by endogenous ethylene.

Effects of carbohydrate and water status on flower opening of cut Madelon roses.

Abstract Flowering stems of ‘Madelon’ roses ( Rosa hybrida L.) severed from the intact plant at the commercially recommended cutting stage and placed in water showed little growth of the corolla and virtually no flower opening. Inclusion of sucrose and an antimicrobial compound in the solution resulted in growth and opening as in unsevered flowers. Flowers placed in water without antimicrobial compounds had a low water potential as a result of vascular blockage in the lowermost segment of the stem. The decrease in water potential was correlated with inhibition of corolla growth and flower opening. The negative effect of water potential on growth and opening was independent of the positive effect of sucrose. Flower opening of ‘Sonia’ roses, cut at the same stage as ‘Madelon’ roses, was as in unsevered flowers. The level of starch in the corolla of ‘Sonia’ roses was twice as high as in ‘Madelon’ roses. ‘Madelon’ roses cut at a later stage of development contained more corolla starch than those cut earlier and both growth and opening were no longer inhibited. The data suggest that the poor opening of cut ‘Madelon’ roses was initially due to relatively low levels of reserve carbohydrates in the corolla while later it was partially due to a low water potential.

Effects of vase-water bacteria on the senescence of cut carnation flowers

Abstract Cut flowering carnation stems (Dianthus caryophyllus L. cvs. Scania and White Sim) were held in water for 7 days at 20°C, after which a low hydraulic conductance and a high number of bacteria were found in the basal 5-cm stem segment. This suggested that bacteria may play a role in determining flower longevity. When taking special precautions, it was possible to keep flowering stems sterile until flower senescence. Petal wilting in the sterile stems occurred at thesame time as in non-sterile controls. Inclusion of antibacterial chemicals in the water also prevented accumulation of bacteria in the solution and in flower stems, but had no effect on flower longevity. Inclusion of bacteria, originating from the vase water of carnation flowers, in vase water at a number that is normally reached after 7 days (107 cfu ml−1) did not significantly hasten flower senescence. It is concluded that the bacterial population developing in the stems of cut carnation flowers during vase life leads to vascular occlusion but this apparently has little effect on flower longevity.

Ultrastructure of autophagy in plant cells: a review.

Just as with yeasts and animal cells, plant cells show several types of autophagy. Microautophagy is the uptake of cellular constituents by the vacuolar membrane. Although microautophagy seems frequent in plants it is not yet fully proven to occur. Macroautophagy occurs farther away from the vacuole. In plants it is performed by autolysosomes, which are considerably different from the autophagosomes found in yeasts and animal cells, as in plants these organelles contain hydrolases from the onset of their formation. Another type of autophagy in plant cells (called mega-autophagy or mega-autolysis) is the massive degradation of the cell at the end of one type of programmed cell death (PCD). Furthermore, evidence has been found for autophagy during degradation of specific proteins, and during the internal degeneration of chloroplasts. This paper gives a brief overview of the present knowledge on the ultrastructure of autophagic processes in plants.

Homologs of genes associated with programmed cell death in animal cells are differentially expressed during senescence of Ipomoea nil petals

In senescent petals of Ipomoea nil, we investigated the expression of genes showing homology to genes involved in animal programmed cell death (PCD). Three encoded proteins were homologous to apoptotic proteins in animals: Bax inhibitor-1 (BI-1), a vacuolar processing enzyme (VPE; homologous to caspases) and a monodehydroascorbate reductase [MDAR; homologous to apoptosis-inducing factor (AIF)]. AIFs harbor an oxidoreductase domain and an apoptotic domain. MDARs exhibit homology to the AIF oxidoreductase domain, not to the apoptotic domain. The three other genes studied relate to autophagy. They encode homologs to vacuolar protein sorting 34 (VPS34) and to the Arabidopsis autophagy-related proteins 4b and 8a (ATG4b and ATG8a). The transcript abundance of MDAR decreased continuously, whereas that of the other genes studies exhibited a transient increase, except ATG4b whose abundance stayed high after an increase. Treatment with ethylene advanced the time to visible petal senescence, and hastened the changes in expression of each of the genes studied. In order to assess the role of VPS34 in petal senescence, we studied the effect of its inhibitor 3-methyladenine (3-MA). 3-MA reduced the time to visible petal senescence, and also accelerated the time to DNA degradation. Remarkably, 3-MA increased the time to nuclear fragmentation, indicating that the time to visible petal senescence was independent of nuclear fragmentation. The data on 3-MA might suggest the idea that autophagy is not a cause of PCD, but part of the remobilization process.

What about the role of autophagy in PCD

Here we discuss to what extent the concept ‘autophagic cell death’ applies to programmed cell death (PCD) in plants. When using the classical definition of ‘autophagic cell death’ the evidence is as yet absent. We examine a possible redefinition of the term.