ProjectMAFF
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MINISTRY OF AGRICULTURE, FISHERIES AND FOODCSG15
Research and Development
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This work addresses the DEFRA objectives of ensuring the reliable supply of high quality produce from competitive UK sources by putting in place underlying knowledge on the post harvest deterioration of ethylene-insensitive cut flowers. Cut flowers are sold on appearance and the ability to retain high quality appearance in the vase adds value to the product. The processes that occur during ageing of the flowers that lead to loss of quality are known as senescence. The senescence of flowers determines the time at which consumers will discard cut flowers and thus affects the value and profitability of the flowers to the grower, wholesaler and retailer. Ethylene is a growth substance i.e. a compound produced naturally in plants that controls specific aspects of their development. Many cut flowers respond to ethylene, by accelerated senescence or petal drop. For these, production of ethylene by the plant, and sensitivity to the plant growth regulator ethylene are the main factors linked to reduced longevity and shelf life and a number of treatments, including chemical and genetic approaches, can be used to increase floral longevity by inactivating the ethylene system. However, for many other commercially important flower species, senescence is not sensitive to ethylene and the techniques presently available are ineffective at prolonging their vase life. Alternative techniques are needed for these ethylene-insensitive plants. Design of these requires detailed knowledge of the physiological, biochemical and molecular biological changes associated with their senescence. With this in mind, the objective of this project was to establish the biochemical and molecular basis of petal deterioration in Alstroemeria. This species was chosen because it is both an important UK crop and, according to the literature, does not show ethylene sensitivity. The project involved collaboration between laboratories at the Universities of Cardiff and Royal Holloway and HRI, Wellesbourne.
Three key areas of the biology of petal deterioration were investigated; protein breakdown, cell membrane biochemistry and cell death. Proteins are important because they direct the biochemistry of the cell, they have structural roles within the
cell and they act as a store of nitrogen. Cell membranes are needed to maintain the integrity of the cell, which is lost during senescence. Senescence leads to cell death, which may be a pre-programmed stage of the life cycle as found in some other biological situations. Initial physiological studies identified seven stages in petal deterioration in Alstroemeria
designated numerically from S0 to S6 and representing 12 days in time. Briefly, S0 and S1 are the stages of floral development when the buds are opening and by S2 the flowers were fully open with the sepals reflexed. At S3 the top three anthers anthesed and two days, later at S4, the petals showed initial signs of in-rolling and discoloration (visible
senescence) while the bottom three anthers had also anthesed. Stage 5 (S5) was defined by the separation of the stigmatic lobes and further signs of petal discoloration and in-rolling. Abscission of the perianth occurred at S6. These stages were used to provide a common framework to co-ordinate investigations in the three laboratories involved in the project.
Protein Breakdown studies: Changes in protein levels and the activity of proteases, the enzymes that cause protein breakdown, were investigated over the duration of the flower vase life. Protein levels fell continuously from the unopened bud, throughout development, until petal abscission while total protease activity in the petals increased more than tenfold between flower opening and S5. Three distinct protease activities could be distinguished and on the basis of studies with specific inhibitors all three were shown to be of the cysteine proteases class. This may be interesting, as cysteine proteases have been found to be associated with cell death in other species. The levels of these activities changed during flower development and senescence with one of the three activities showing an increase. Studies on the expression of an Alstroemeria cysteine protease gene sequence revealed that the gene was expressed from the earliest stages analysed but a dramatic increase took place as the petals aged.
A well-known mechanism for protein degradation involves attaching ubiquitin, itself a protein, to the protein destined for breakdown. Ubiquitin acts as a label and targets the degradation process to the attached protein. In Alstroemeria, no important changes were detected in the expression of the ubiquitin gene or ubiquinated proteins throughout petal development and senescence. These results suggest that, in contrast to other lilies such as Hemerocallis, ubiquitination may not be an important mechanism controlling protein degradation in this species.
Membrane integrity and lipid metabolism: The project has provided new insights into lipid and membrane breakdown during flower ageing. During senescence, most of the lipids in the membrane were lost through metabolism. As the membranes aged and were depleted, their integrity was progressively lost, as measured by leakage of ions. Lipid peroxidation had previously been hypothesised as the decisive factor in membrane deterioration, but although it was detected, its importance was less than in some other flower species and lipoyxgenases also had a lesser role. Oxidative damage may have played a part in flower deterioration as the levels of carotenoids, which are antioxidants were higher in petals than sepals and this was correlated with reduced damage in petal tissues.
Cell death: The involvement of programmedcell death in Alstroemeria petals was tested for by three standard methods. DNA laddering was used to reveal patterns of DNA fragmentation which have been found in other systems to be a hallmark of programmed cell death. Laddering was evident in Alstroemeria petals even in young petals although more extensive laddering was detectable during petal deterioration. The second approach was to look for genes that act in other systems that act as markers of cell death. One of these, DAD1 (defender against apoptotic death) is down-regulated as cells enter cell death in most systems studied and a homologue of DAD1 was isolated from Alstroemeria as a partial petal cDNA clone. The expression pattern of this gene showed a sharp decrease at stage 4 and hence may provide a useful marker for this stage in petal deterioration. A further method, the TUNEL assay for DNA breakdown was unsuccessful in this project.
Major conclusions and suggestions for further work: The major overall research finding is that there is no simple triggering event for petal senescence in Alstroemeria, as in ethylene sensitive species, but petal ageing represents a gradual deterioration. A number of genes and processes were identified as changing concurrently in ageing flowers. Diagnostics for post harvest quality therefore will need to be based on simultaneous monitoring of multiple components. An additional finding is that some of the processes activated during petal senescence may already be in place while the petals are still developing. This is significant as it indicates that it may be possible to develop diagnostic approaches for flowers at the stage of picking as well as later in their shelf life. It also suggests that it will be essential to understand the effect of treatments during packaging and shipping of cut flowers on the senescence process.
Besides contributing to DEFRA objectives of ensuring the reliable supply of high quality produce from competitive UK sources these results provide the foundation and guidance to their further achievement through the application of appropriate (post-genomic) technologies. We propose the next steps to build on the existing work.
1Develop DNA microarray chips for diagnostic studies on flower deterioration
2Use these diagnostics to identify sensitive points in the supply, storage and distribution chain.
This project has provided important new information and understanding of the factors involved in deterioration of ethylene insensitive cut flowers. This will underpin the competitive position of UK Horticulture in the growing market for cut flowers. The information has been disseminated through peer reviewed and horticultural trade publications and at
national and international conferences. UK industry has confirmed the relevance of the work by supporting proposals to follow the work up via BBSRC and DEFRA.
Objectives and relative milestones which were changed during the course of the project and Reasons for making the changes:
Milestone number / Original milestone / New milestone / Reason for change02/05 / Northern analysis of delta-9 desaturase and LOX genes / Northern analysis of Alstroemeria LOX genes / Work on rose was not continued and delta-9-desaturase was less relevant to Alstroemeria
S03/02/01 / Preparation of probes to genes coding for enzymes involved in the ligation of ubiquitin to proteins / comparison of methods of lipid hydroperoxide quantification / Results from transcriptional and western analysis of ubiquitin expression suggests that ubiquitination may not be a major player in this system
S02/04/02 / Assess GST levels using CDNB substrate / Antioxidant status of sepals and petals / Antioxidant studies were considered to be
S02/04/03 / Determination of further substrates for GST assay / Petal and sepal pigment levels, including total carotenoids, chlorophyll and anthocyanins / more relevant to the emerging picture
S02/05/01 / Assay delta9 desaturase using radiolabelling / Removed / Work on rose was not continued and delta-
S02/05/02 / Preparation of probes for analysis of delta 9 desaturase and LOX / Preparation of probes for analysis of LOX expression / 9-desaturase was less relevant to Alstroemeria
At an early stage in the project it was agreed with the PMO that efforts would be concentrated on Alstroemeria and thus work on rose varieties would not be undertaken. This decision was based on difficulties in finding a suitable variety for the work, and an appreciation that petal senescence in lileaceous species is of greater scientific and commercial relevance.
CSG7 objectives and to what extent they were met
Objective / Title / Objective fully met?01 / Establish a common set of parameters for assessment of wilting in the two species.
Including fresh weight, surface area appearance etc. / yes
02 / Investigate membrane integrity during petal senescence:
02/01 / Undertake measurements of conductivity and/or osmolarity and factors affecting cell membrane permeability (Y1) / yes
02/02 / Quantification and identification of membrane lipids (Y1) / yes
02/03 / Activity of enzymes involved in lipid peroxidation (Y2) / yes
02/04 / [Activity of GSTs using a range of suitable substrates substituted with analysis of antioxidant status ] (Y2) / abandoned in favour of important studies on petal antioxidant status
02/05 / Northern analysis for timing expression of related genes: LOX and [(in roses) delta-9-desaturase removed 31/08/99.(Y1-2)] / yes for LOX expression in Alstroemeria, using alternative method
03 / Protein degradation during petal senescence:
03/01 / Western and northern analysis for protein degradation via the ubiquitin pathway and ubiquitin gene expression(Y1) / yes, using alternative method, although the results were not very informative
S03/01/02 / Determine overall protein changes using SDS PAGE (Y1) / yes
03/02 / [Using probes to genes encoding enzymes in the ubiquitin pathway, follow their expression:substituted with comparison of methods of lipid hydroperoxide quantification] (Y3) / abandoned due to the nature of the results from 03/01. New objective was met in full
03/03 / Using probes to cysteine proteases, determine the timing of expression of these genes in relation to loss of protein. (Y1-2) / Yes, using an alternative method for expression analysis
04 / Charting cell death during petal senescence:
04/01 / Using TUNEL, investigate levels of DNA fragmentation (Y3) / Attempted but not successful
04/02 / Assess levels of DNA laddering (Y3) / yes
04/03 / Use northern analysis to investigate expression of the dad1 gene (a key control gene for cell death) (Y3) / Yes, using an alternative method for expression analysis
05 / Technology transfer and communication of results:
05/01 / Preparation of papers for publication in refereed journals in the three major areas of research (Y2-3) / Yes (see Annex)
05/02 / Preparation of article for horticultural trade press (Y3) / Yes (see Annex)
05/03 / Presentation at Grower Conference(s) (Y2-3) / Yes (see Annex)
Introduction
The senescence of flowers determines the time at which consumers will discard cut flowers and, in consequence, affects the value and profitability of the flowers to the grower, wholesaler and retailer. The production of cut flowers in the UK is worth in excess of £100m pa compared with an import value of over £220m (MAFF statistics, 1995 data). Increased quality and shelf-life of flowers would be of considerable economic benefit to producers (increased crop value) and retailers (increased shelf life) as well as the consumer (increased vase life). In order to bring about such improvements a thorough understanding of the biochemical and physiological processes which lead to the deterioration of the products is required. Research to date has identified that the plant growth regulator ethylene is intimately linked with the reduced longevity and shelf life of some flowers. For these species, treatments, including chemical and molecular means, can be used to increase floral longevity. However, senescence of many economically important species is not ethylene sensitive and thus the techniques presently available are ineffectual at prolonging their vase life. As yet, there is little or no information on either the controlling mechanisms which initiate the senescence of these tissues (van Doorn and Stead, 1995), or the biochemical events which occur during the senescence process. Understanding the molecular and physiological changes associated with senescence will allow a targeted approach to this problem. Alstroemeria was chosen for the subject of this study as it is both an important UK crop (sales increased by 35% in 1998 relative to 1997, Flower Business International, Feb, 1999) and, according to the literature (Woltering and van Doorn, 1988), shows little or no ethylene sensitivity.
The primary purpose of this work, therefore, was to gain an understanding of the processes occurring during the degeneration of ethylene-insensitive flowers. Three key areas were chosen for study: changes in membrane composition, protein degradation and processes associated with cell death. These processes were chosen as all three have been associated with senescence and were likely to provide information on the timing of biochemical and molecular changes occurring in Alstroemeria petal development and senescence. Protein degradation is important both globally in the re-mobilisation of nutrients from the senescing tissue, but also degradation of specific proteins and activation of specific proteases are key control elements in other systems (Glotzer et al, 1991; Lam et al, 1999). Membrane integrity is an essential component of living cells and is lost during senescence, thus investigation of this process was also considered to be important in charting petal wilting in Alstroemeria. Cell death is the final
process in terminal senescence and the timing of its onset indicates that the final phase in this developmental process has been reached.
(1) Membrane damage
The process of lipid peroxidation (the addition of oxygen to membrane unsaturated fatty acids) is an important event associated with senescence (Lesham, 1992). In some tissues this appears to be a late phenomenon occurring coincidentally with cellular death and
may, therefore, be a consequence rather than a cause of the loss of membrane integrity. However, in other cases, lipid peroxidation precedes any obvious signs of senescence and therefore appears to be a primary event associated with the onset of senescence (Zhuang et al, 1995). In either case, the initial changes in lipid composition are likely to be associated with the deacylation of the membrane lipid components through the action of phospholipases. The released fatty acids then serve as substrates for lipoxygenase (LOX) mediated oxygenation and/or autoxidation (ie nonenzyme catalysed reactions) processes.
The role of enzymes that protect against membrane damage such as glutathione-S transferases (GSTs) was also investigated Understanding the role of GSTs and timing of their expression during petal senescence may be important in understanding the processes leading to irreversible membrane damage. GST encoding genes have been associated with petal senescence in other species (Itzhaki and Woodson, 1993) and may be important in delaying membrane damage while remobilisation of nutrients takes place. This is achieved by conjugating glutathione to cytotoxic compounds produced during membrane breakdown, resulting in their detoxification (Marrs, 1996).
(2) Protein breakdown
The ability to selectively degrade proteins is essential in all tissues, therefore changes in the pattern of protein degradation would be expected during both flower development and senescence. Moreover, the loss of membrane permeability seen during petal senescence may be the result of the selective degradation of integral membrane proteins. One way in which this may be achieved is to target specific proteins by conjugating ubiquitin moieties to the target protein, the ubiquitinated protein is then degraded by an ATP-dependent protease (Callis, 1995).
In the ethylene-insensitive flowers of Hemerocallis patterns of protein ubiquitination clearly alter suggesting that specific proteins are targeted for degradation in this way. Furthermore, treatment with cylcoheximide (CHI - an inhibitor of translation) delayed flower senescence in daylilies (Courtney et al., 1994) and iris (van Doorn et al, 1995), whilst senescence was also delayed after CHI treatment in the ethylene-sensitive petal of carnation (Wulster et al, 1982a,b). In daylily several different ubiquitin transcripts were detected by northern analysis which were differentially expressed during flower development and senescence and CHI treatment resulted in the accumulation of several of these, however one transcript was down regulated (Courtney et al, 1994). The identification of specific proteins which become ubiquitinated prior to visual signs of petal wilting will assist in the characterisation of critical events controlling the wilting process.