Hello Evangelia,

the best method is to compare the dry weight and the fresh weight before and after, when you see death of leaves; you can repeat this method, until you see total death of plant.

Cordially,

Houssein

Hello Houssein.

Thank you for your response. However this method would be destructive. I sould have mentioned it in my discription that I would rather prefer a non-destructive one.

hello

I suggest you use the LI6400 to measure the Photosynthetic parameters, or the Chlorophyll meter for Chlorophyll determination, then show you the senescence process. The photosynthetic decrease rate and the degradation rate of Chlorophyll may be able to represent the leaf death rate. Best wish for u!

As a non-destructive method I would suggest PAM-imaging of plant leaf (chlorophyll fluorescence), Photosynthetic efficiency by photosynthetic efficiency analyser, or chlorophyl content by a Portable chlorophyll meter (there are many options for that non-destructive meter). I think PAM-imaging chlorophyll is the best but depend on the instrument availability..

Thank you Abdelghafar Abu-Elsaoud.

I do intend to perform some of these physiological measurements, but I was wondering if there was a method to access the plants' phenotype. % of leaf senescence etc.

May be you can use image analysis using ImageJ ( http://rsbweb.nih.gov/ij/ ) or other image processing software. With a series of images from a fixed angle and fixed distance from plants and with a ruler, you can calculate different different quantitative parameter.

as example:-

http://www.plantmethods.com/content/8/1/45

http://www.ncbi.nlm.nih.gov/pubmed/22183684

http://link.springer.com/protocol/10.1007%2F978-1-61779-501-5_41

There is not a single answer and a single "best method". It is the species and, therefore, its growth form, possibly its branching pattern, that may allow for an access via the plant´s phenotype: If you are able to detect ages of branch sections (as is the case e.g. in many conifers or eucalypts) the number of remaining leaves on each branch´s age class allows for determination of "death" rates via mathematics used in population biology. Another direct way is to mark leaves and directly observe leaf loss from the marked population over extended periods. However, it´s the species that determines the approach to be chosen.

Measurements via leaf gas exchange etc. may help to follow the aging as such but not to determine a death rate (e.g. Ecotropica 10(2) page 103 (2004) or Australian J Plant Physiol 15 page 292 (1988) and lots of literature from the early to mid 1980s). What are the species you are working with?

You may think about quantifying the leaf area changing colour (e.g. in maple laves).... as a measure of senescence. However, this is not a death rate....

Interesting debate. For a single leaf there is no "rate of death". The leaf is either alive or dead. If you mean "rate of senescence" then the rate of chlorophyll breakdown (by wet chemistry or remote sensing) is a concensus estimate. Surprisingly, unlike the animal kingdom or humans, we still do not have a concensus definition and measurement of plant or single leaf death. We have difinitions of cell death (apoptosis). Photosynthesis is not a useful symptom of plant death. It can stop under stress but then recover after stress removal. Or it can terminate in older leaves while still proceeding in the younger ones. For intact leaves under drought stress, the leaf water potential from which a leaf cannot recover in terms of water status is the point of leaf death. The natual death in plants (probably linked to reproduction) should be easier to define.

Dear Manfred Kupper,

Thank you for your reply. I am working on Miscanthus physiology under abiotic stresses. The reason for the explanation under my question was mainly because the initial responses I got referred to physiological measurements which is in one hand indirectly relevant to the rate of leaf death yet not exactly what I was looking for. In brief I I was wondering what protocol or methodology to follow in order to measure leaf loss in each plant in each stress treatment. For example the frequency of keeping track of leaf loss during the determined experimental period...etc.

Dear Abraham Blum,

Thank you for your response. I truly appreciate it and it is an honor. I agree with everything you've said and allow me to think of you as a mentor as I have read thoroughly your work. However, I do mean leaf death and not leaf senescence as at least for salinity it is related to the rate at which salts accumulate to toxic levels (Munns et al.,1995).

Evangelia,

Do you mean a protocol to measure the rate at which leaves die? That depends on the previous, broad knowledge you have of the effects of the stress. I mean, deoending on the kind and duration of the stress you will have the first leaf dying after X days and then probably some sigmoidal function of dead leaves accumulation, so you will need to adjust the frequency of measurements to the critical phases of death onset, linear increase, saturation. If you have no idea of that and you have the possibility, first perform some simple assays to determine the general response and then repeat in a more elaborated way.

A good approximation for a general survey would be to take general pictures of the plant and quantify the % dead area: I assume this can be seen as a change of color of the leaf or any other external sign, or you search for a nondestructive method would make no sense as you are already taking physiological measurements

The rate of litter fall is used in ecology to measure LAI in difficult situations. There is a regression between living crowns and the amount that drops off. Several (10+) squared buckets made of of ironwire are located under the canopy using a random coordinate generator. At given intervals the buckets are cleared and the content measured. The area proportion of bucket footprint is then related to total area.. journal of functional ecology J Roberts, vol 27 2002 and many others. Leaf death intensity and time can be analyzed with time series tools in order to detect synchronies or unusual trends. I hope

Evangelia,

You have an interesting case! It's good to see Abe Blum contributing his years of experience to the discussion, though as he says, it is difficult to define what is meant by leaf death. Senescence itself is an active process with a series of genes being switched on once the plant has decided that a particular organ is no longer needed. Therefore, it is impossible to monitor the rate of death of leaves (at least while they are still metabolically active). You can monitor their rate of senescence, and many contributors have suggested non-desctructive options for this.

If you are wanting to monitor rate of senescence in a canopy and don't have sophisticated imaging technology for this, a simple digital camera can be used, placed on a tripod in a constant position, using readily available software to count pixels in red, green and blue channels and to quantify the proportion of green present in the image. I have done this myself. I remember seeing a publication on this some 15-20 or so years ago, but can't now remember where!

Although you probably won't want to do the following as it is a destructive method, but the induction of senescence-specific promoter sequences could be used to identify when the (irreversible) process of senescence of a particular organ has been initiated.

Back in the 1980s a colleague of mine at the John Innes Centre (Tristan Dyer) identified and transformed tobacco with a senescence-specific promoter attached to a gene for cytokinin production, so that cytokinin production was stimulated as soon as senescence was initiated. The result was a tobacco plant with very delayed loss of chlorophyll.

All the best with your Miscanthus studies in balmy Aberystwyth!

Steve

As a PS, the paper I was thinking of which used a digital camera to measure leaf senescence might have been this one: Crop Science (1999) 39:719-724, which appears to be available at http://naldc.nal.usda.gov/download/50473/PDF.

Steve

Thank you Steve this is very interesting and actually it is in my plans to do image analysis for quantifying the % of dead area for each of my treatments. All this discussion on the topic gave me so many nice ideas and food for thought.

As Xavier suggested and it was what I was looking for, I want to measure the rate at which leaves die. So, I had in my mind to measure the date when the first leaf dies and then the rest of the leaves and try to fit a curve/build a model. The only think is that I don't know how often I need to check for dead leaves. Do I check every day and see how it goes or do I set frequent time points? I guess I just need to try it and see how is going.

Hi Evangelia,

OK, assuming you want to measure the proportion (percentage) of dead leaves, until you know how rapidly they will become dead (dry and brown), you won't be able to decide how frequently you will need to monitor them. At least, if you start off measuring frequently (every day?) until you know what time period makes sense, you won't have missed anything interesting.

Appearance of dead leaves is going to depend on many factors (pot size, nutrient availability, light intensity and duration, plant density, water availability, soil characteristics, temperature, genetic predisposition to shedding lower leaves, and so on), so unless you have already done a pilot study to get some idea of rate of death, you will just have "to try it and see how it is going".

.... one thing about Miscanthus - I've never grown it, but I am assuming it will remain vegetative during the course of your experiments. If not; if parts of the plant become reproductive, that may have an effect on the rate of death of lower leaves, as a result of the plant suddenly diverting its resources into reproductive development.

All the best with it.

Steve

Adding to Steve proposal on senescence-specific promoters, maybe you can afford some degree of destruction using leaf discs taken with a cork borer: for each leaf you sample, most of the leaf will still be there as (I assume) you do not need much leaf material to identify those promoters and Miscanthus leaves are quite long

Ok, if the objective is the % of dead leaves, one question: do you really need % of dead LEAVES or % of dead leaf AREA would do? The latter is much easier to determine through image analysis, and for a pilot study it would be very simple to take a picture each day, fit a curve, and then see which are the critical points, depending on the form of the curve. I would bet for a sigmoidal, so you would need onset of leaf death, point of change to linear phase, end of linear phase (this will give you the inflexion point, around half way). Then, even if you really need to record the death of each individual leaf (much more work), the former would give you a clue on how often to take measurements, just for leaf death or for any other parameter you intend to measure.

Regards

I did not catch you were doing Miscanthus (the hybrid giganteus is sterile) in pots. Also death is probably leaf fall (phylloptosis?). I remain stuck to the simple idea of a net bag around plants and to time series applications on leaf area duration. Also leaf area density should sense stress, but as far timings, in the open M. should drop leaves in winter and all senescence cycle is tempered by temperature. Frequently in canes leaf detachement is aided by hand removal with moderate pressure. After each treatment the number of naked nodes would quantify the progress in 'death. In greenhouse there will be a transition to longer leaf cycles and the problem is the real relevance to cultivation. Senescence is progressive as basically canes are evergreens.

Yield still wonder whether there is really any direct measure to account for leaf death. Leaf death is a slow process just as ageing is. Yet, there can be four different methods, although one may not agree with me:

1 slow and steady loss of chlorophyll leading to chlorosis

2 Formation of abscission layer

3 phloem unloading (Down movement of photosynthates)

4 Measurement of Hill reaction ( PS II )

An integrated approach of all these should give a measurement leaf health/leaf death

One such measure is the senescence percentage

one of the methods is scaling the senescing area. make a scale 1-5 from the smallest area which is possible to appear and the largest that might be the death leaf. make a gradient to fulfill the scale between 1-5.