Petal Senescence: New Concepts for Ageing Cells

Senescence in flower petals can be regarded as a form of programmed cell death (PCD), being a process where cells or tissues are broken down in an orderly and predictable manner, whereby nutrients are re-used by other cells, tissues or plant parts. The process of petal senescence shows many similarities to autophagic PCD in animal cells including a massive breakdown of protein, DNA and RNA, the formation of autophagic vacuoles for the breakdown of cytoplasm and organelles therein and, the eventual rupture of these vacuoles that kills the cell. Chromatin condensation and DNA and nuclear fragmentation (traditionally considered being apoptotic-like features) are observed in both autophagic animal cells and in senescing petal cells. We present a conceptional model underlying petal senescence that integrates elements that have been associated with both apoptotic and autophagic types of PC

Vascular occlusion in stems of cut rose flowers

The quality of cut rose flowers, a major horticultural crop in the Netherlands, is often unsatisfactory. During vase-life premature signs of water stress occur, such as slow growth of the bud which often results in poor flower opening, wilting of both the flowers and the leaves, and bending of the stem just underneath the flower. These symptoms are due to an inability to take up adequate amounts of water from the vase solution, which in turn is due to an occlusion in the lower part of the stem. Experiments in which a razor blade was introduced into the stem showed that more than 66% of all xylem conduits must become non-functional before a reduction in the rate of water uptake becomes apparent. This study distinguishes between the occlusion which occurs when the stems are placed in water immediately after harvest, and the occlusion which occurs as a result of dry storage.When the stems are placed in water directly after harvest the blockage could be due to processes inherent in the stem, e.g. as a result of a wound-reaction, in which ethylene is generally involved. Alternatively, it could be due to microbial growth. Inhibition of the production or action of ethylene had no effect on the blockage. Light- and electron microscopy revealed no tyloses in the lumen of the xylem conduits (vessels and tracheids) and in only a few conduits a deposit of gummy material occurred in the absence of bacteria. This material stained with ruthenium red, a dye on polysaccharides. Slime on the colonies of bacteria isolated from the stems also stained with ruthenium red, indicating that the material found in some conduits might be bacterial polysaccharide that was able to pass the pit membranes between the conduits. These membranes contain small pores, through which the water flows from one conduit to the next. During vase-life a population of bacteria developed on the cut surface and inside the opened xylem conduits. The bacteria were always accompanied by extracellular polysaccharides. Fungi were also observed at the cut surface, but only after the occlusion had already occurred, and yeasts were not observed. When bacterial growth was excluded the blockage was absent. It was concluded, therefore, that the blockage occurring in the stems that are placed in water directly after harvest is caused by bacteria.Isolated living or dead bacteria both resulted in vascular blockage, when given either at room temperature or at 1°C This indicates that physiological activity, either from the part of the bacteria or from the part of the stem, is not a prerequisite in the bacterial blockage. Isolated bacterial polysaccharides and proteins such as cellulase and the inert ovalbumin, both with a molecular mass of about 50 kilodaltons, also resulted in rapid blockage. It is concluded, therefore, that the occlusion is due to a purely physical effect of living bacteria with their extracellular polysaccharides, as well as dead bacteria and their degradation products. The blockage is probably mainly due to the obstruction of the passage of water at the pit membranes between the xylem conduits.The effects of dry storage can also be partially microbial in origin, as the number of bacteria associated with the cut surface and the xylem interior increase during dry storage. When the stems are not temporarily placed in water after harvest, however, they do not become contaminated with bacteria. When such stems are held dry an occlusion also develops. Among rose cultivars great differences were found in the time-course of this blockage. In Cara mia roses, for example, it occurred within 3-4 h (at 20°C), in Madelon within 9-14 h, in Sonia after 24-36 h, and in Frisco after about 48 h of dry storage. The last cultivar apparently conserves water as its stomates often closed more rapidly, and its rate of cuticular transpiration was lower. However, in Cara mia, Madelon, and Sonia roses stomatal response, water loss, and water potential during dry storage, showed the same time course.The blockage that is not microbial in origin could be due to processes in the stem, for instance as a response to the low water potential. A microscopical investigation, however, did not show evidence for any material, be it tyloses, amorphous plugs or hydrophobic substances, in the xylem lumen.After cutting air absorption occurs at the cut surface, due to the receding water columns in the xylem conduits opened by cutting. This absorption ceased already within half an hour. In the absence of a leaf nearby the cut surface the amount of absorbed air corresponded with the volume of the lumen of the opened conduits. The occlusion related to dry storage, however, occurs only after three hours in the most sensitive cultivar tested. It was concluded, therefore, that the mere presence of air in the lumen of the conduits opened by cutting is no obstacle to the subsequent flow of water.Three hypotheses were tested as to the origin of the blockage due to dry storage:Firstly, when the lumen of the conduits that are opened by cutting is blocked by the presence of air, the water might follow the xylem walls until reaching non-opened conduits filled with water. When the walls would dehydrate this second pathway could become inoperative. This hypothesis was tested using rose stems of which the cut surface was covered with laboratory grease, and a pan of the bark higher on the stem was cut away (girdling), thus exposing cell walls adjacent to the xylem. Girdled Sonia rose stems which were placed in water without dry storage remained turgid, provided that the wall area in contact with water was more than 0.6 cm 2. When the girdled stems were held dry and then placed in water the uptake of water was strongly inhibited, with a time course reminiscent of the blockage in normal stems. These results support the above hypothesis. When the dry-stored girdled stems were placed in a surfactant solution, however, the rate of water uptake was not improved, whereas the rate of uptake was greatly improved in dry-stored non-girdled stems. It was concluded, therefore, that a reduced access via the cell wall pathway is not the cause of the occlusion.Secondly, water might initially be able to partially compress the embolus in the conduits opened by cutting, thereby providing contact with adjacent conduits filled with water. An occlusion would occur only when the walls dehydrate to the extent that water, because of its surface tension, is no longer able to contact these walls. This hypothesis was tested by placing stems in a suspension of india ink for 1-5 h, after dry storage. After 5-180 min of dry storage water was still able to partially penetrate the conduits of Sonia roses, and even after 24 h this was true, although the penetration depth was smaller after 24 h as compared to 3 h. When the stems were placed in a surfactant solution after 24 h of dry storage the penetration depth of the water was much higher than after 5-180 min of dry storage, but not as high as in controls that were not stored dry. These results are in agreement with the hypothesis. In Cara mia roses, however, the results were the same as in Sonia. As the occlusion occurs already within 180 min in the former cultivar, it is not correlated in time with a reduction of the penetration depth of water in the conduits opened by cutting.Thirdly, the occlusion may be related to cavitation of the conduits that am not opened by cutting. Cavitation is the sudden filling with gas of a liquid-filled conduit; it may occur spontaneously when the water potential becomes low, or as a result of pulling gas from an adjacent conduit that is already gas- filled. Cavitation results in ultrasonic acoustic emissions (UAEs) which can be detected by placing a microphone at the surface of the stem. The frequency of UAEs always increased prior to the development of the occlusion, in Cara mia, Madelon, and Sonia roses. As these cultivars show a different time until the occlusion occurs, the results suggest that cavitation is an important cause of the occlusion. In Cara mia roses cavitation appears to be the only cause of the occlusion, in the other cultivars tested the incapacity of water to penetrate the conduits opened by cutting may also be part of the blockage

Effect of precooling and ethylene absorbent on the quality of Dendrobium "Pompadour" flowers

We studied the effect of precooling and the use of an ethylene absorbent (based on potassium permanganate) in the flower boxes, on the vase life of Dendrobium `Pompadour` flowers, after simulation of air shipment (3 days at 25°C). Precooling at 10°C (85-95%RH) for 60 minutes reduced ethylene production, ACC activity, and the concentration of 1-aminocyclopropane-1-carboxylic acid (ACC) in the flowers, during shipment. Precooling for 90 minutes or longer did not have a positive effect on the chilling-sensitive Dendrobium flowers. The presence of an ethylene absorbent in the cardboard boxes further reduced ethylene concentration in the boxes. The combination of 60 min precooling and the ethylene absorbent was optimal to reduce epinasty of the buds and flowers, to promote bud opening and to prevent abscission of open flowers. It also considerably delayed the time to in visible petal withering

Snijbloemen : kwaliteitsbehoud in de afzetketen

Behandeling van fysiologische, technische en organisatorische factoren die van invloed zijn op de kwaliteit van snijbloeme

Gene expression in opening and senescing petals of morning glory (Ipomoea nil) flowers

We isolated several senescence-associated genes (SAGs) from the petals of morning glory (Ipomoea nil) flowers, with the aim of furthering our understanding of programmed cell death. Samples were taken from the closed bud stage to advanced visible senescence. Actinomycin D, an inhibitor of transcription, if given prior to 4 h after opening, suppressed the onset of visible senescence, which occurred at about 9 h after flower opening. The isolated genes all showed upregulation. Two cell-wall related genes were upregulated early, one encoding an extensin and one a caffeoyl-CoA-3-O-methyltransferase, involved in lignin production. A pectinacetylesterase was upregulated after flower opening and might be involved in cell-wall degradation. Some identified genes showed high homology with published SAGs possibly involved in remobilisation processes: an alcohol dehydrogenase and three cysteine proteases. One transcript encoded a leucine-rich repeat receptor protein kinase, putatively involved in signal transduction. Another transcript encoded a 14-3-3 protein, also a protein kinase. Two genes have apparently not been associated previously with senescence: the first encoded a putative SEC14, which is required for Golgi vesicle transport, the second was a putative ataxin-2, which has been related to RNA metabolism. Induction of the latter has been shown to result in cell death in yeast, due to defects in actin filament formation. The possible roles of these genes in programmed cell death are discussed

Literatuurstudie naar de moleculaire kennis rond de balans tussen vegetatieve-generatieve groei van aardbeiplanten

De situatie waarbij de aanleg van blad en bloem in goed evenwicht is, zodat de gewenste fysiologische en productieve stadia van de plant bereikt worden, is van groot belang voor de aardbeien sector. In opdracht van het Productschap Tuinbouw en onder begeleiding en advies van Plantum is een literatuurstudie uitgevoerd naar de moleculaire kennis op dit gebied bij planten in het algemeen en de vertaling ervan naar meerjarige aardbei planten. Recent onderzoek op dit gebied heeft geleid tot de identificatie van een aantal sleutelgenen die de regulatie van bloemaanleg en ontwikkeling bepalen

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 Ipomoea nil (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

Comparison of micro-array profiling in senescing iris and carnation flowers

Gene expression profiles of cut Iris and carnation flowers were studied using cDNA microarrays. The cDNA libraries were enriched for flower-specific genes by subtraction with cDNA from subtending growing tissue. This strategy is meant to eliminate most household genes and numerous genes that are not specific for petals and senescence. In Iris, we spotted about 1400 clones and in carnation about 2000, of which 220 and 90 clones respectively were (partially) sequenced. Unexpectedly, during Iris senescence up-regulation was observed for many genes that previously had been characterized as being defence-related. Although such genes were also found in carnation, their relative contribution to the changes in expression seemed less pronounced. Another remarkable result was the limited number of known ethylene-related genes in carnation that were detected. Among those found was ACO1. Other ethylene-related genes may have been lost in the subtraction; and ACO1 seems specific for the ethylene climacteric. No ethylene-related genes were found in Iris. Since ethylene does not regulate petal senescence in Iris this is no surprise. Some similarities were found between Iris and carnation. In both species a considerable proportion of the up-regulated genes encode enzymes that are involved in the degradation of lipids, protein, and complex carbohydrates such as cell walls. Several genes involved in signal transduction and in transcription were observed to change expression levels in both species, but none were the same in both species, as judged from the limited sequence information. A novel EIN3 (EIL) transcription factor was discovered in carnation. The expression pattern of some putative transcription factors in carnation were expressed independently of ethylene treatment, and may be candidates for early regulators of traits such as ethylene senstivity. The detailed results on Iris have been published in the December 2003 issue of Plant Molecular Biology (53: 845-865); the results on carnation have been submitted