Chlorophyll fluorescence appears to be a measure of photosynthesis, but this is an over-simplification. Fluorescence can measure the efficiency of PSII photochemistry, which can be used to estimate the rate of linear electron transport by multiplying by the light intensity. However, researchers generally mean carbon fixation when they refer to photosynthesis. Electron transport and CO2 fixation can correlate well, but may not correlate in the field due to processes such as photorespiration, nitrogen metabolism and the Mehler reaction.To use measurements of chlorophyll fluorescence to analyse photosynthesis, researchers must distinguish between photochemical quenching and non-photochemical quenching (heat dissipation). Please have a look at the enclosed PDFs..

Dear Prof. Srivastava

Thanks for your response.

Please read my question again. I have asked the TIME of Chlorophyll Fluorescence measurement from plants growing in open field and in the shade???

Meanwhile, the 2nd pdf file is irrelevant to my question.

Thanks for your reply and help.

The time doesn't matter. It is the process that is important. You expose the leaf to a defined wavelength of light and measure the amount of light re-emitted at a longer wavelength. So you can do this at any time you like :)

You should wrap in aluminium foil apical leaves and then induce a transition to light following the creation of quenchings by a PAM fluorimeter. All this makes sense if some stress have occured on plants over night like low temperature, salinity or water shortage. Also ontogeny and leaf age may chnage the signal but usually the photosystems are the last reaction to a stress. See the topic under photoinhibition and chl fluorescence.

Dear Mr. Hussain,

For planning your experiment, specifically, the time at which you would like to make the fluorescence sampling, well, the answer depends actually on several factors:

Are you planning to make a diurnal changes experiment? You want to observe how the photosynthetic machinery of your plants works along a full sunny day? or along a full week or a full month? Then you will have to measure periodically, every hour? every two hours? Do you want to establish a relationship between stomatal aperture and primary photochemistry events monitored with Chl a fluorescence? are you following the evolution of drought stress in a big or small field? What exactly are you trying to observe? The precise time of the day, it's important for you? why? Do you need only one single measurement per day? why? If you need to measure along a full day, then the interval in between the diurnal variations measurements depends on the the time scale resolution you want to have. Do you want to observe rapid changes after application of a specific stressor (herbicide, heavy metal, heat, cold, salt, any other plant stressing condition) or simply you want to observe diurnal photosynthetic behavior of your plants? Are those changes expected to occur fast or it can takes several hours to evolve? OK, let's say, you make a diurnal changes sampling for studying diurnal variations in leaf temperature, stomatal aperture, chlorophyll fluorescence, gas exchange photosynthesis, protein expression, induced levels of abcisic acid, or any, and you start at 6:00 AM, preparing a tea or a coffee and your plants for dark adaptation, and after 30 minutes you are ready for measuring fluorescence and depending upon the amount of plants to measure and the time it takes for each measurement, then you could run your first sampling campaign of the day for about 30 minutes to 1 hour... or more... how many plants do you have to measure? How long are you going to invest in measuring each plant? How many leaves are you going to measure in one plant? how easy it is to reach the leaves? What about sampling the other parameters? In a word, do you have a clearly defined measuring protocol? have you already done a series of measurements for knowing how long does it take to sample fluorescence in one leaf? in a whole plant? in one full plant, shrub, bush? Is one leaf from one plant enough representative for you? are the leaves enough big to measure? to sample a full plant (if several leaves are going to be measured), how many plants are you going to measure? You need to know all that before planning an experiment, any experiment, in order to optimize sampling time, and the experiment itself. OK, let's say, you plan to make a diurnal changes experiment to observe how the plant behaves along a full day, and you start your sampling campaign, now once that you have collected your fist set of data, can you immediately transfer all the collected data to your portable computer? or do you have to go back to the lab to do that? How long does it takes to transfer the data? can it wait until you have finished all of your measurements? Does your Fluorometer machine has enough memory to keep all of the measured data in it until the end of the day? That takes time also. And here is another important question to answer> are you going to measure ONLY fluorescence or do you plan to measure other parameters? Do you plan to measure plant spectral reflectance, chlorophyll content, CO2/O2 evolution photosynthesis, or any other kind of measurements beside fluorescence? OK, let's assume you collect and prepare next sampling in a short time, 30 minutes for example, remember that you need to put your plants in darkness (dark adaptation for at least 30 minutes) before measuring Chlorophyll a fluorescence. What it is your own personal time availability? Do you have to attend lectures in between? Do you have technical staff in charge of the experimental sampling ?  And another important question to answer is: In order to have comparable data, you need to optimize the speed of sampling time, the amount of plant individuals to measure. it has to be as closest as possible in time. So, many questions to be answered before you can get the optimal answer to the one simple question. In a word, the best moment of the day to measure fluorescence depends upon many factors.

For comparing the photosynthetic behavior of two groups ornamental shrubs same species same variety, growing in two different light regimes: in shade and in open light conditions, and assuming any other conditions are kept the same (soil humidity, soil chemistry, wind speed, atmosphere chemistry, etc), I would recommend a diurnal changes experiment.

So, you measure Chl fluorescence every 2 hours from 6 AM until 8 PM. Each 2 hours, you should measure also (at least) : ambient light, air temperature, soil temperature, soil pH, soil humidity. You may repeat this measurements, once in a full sunny day  and once in a cloudy day. You could repeat it also along once per week during several weeks. Preferably, take your measurements before flowering, as flowering introduce a lot of "noise" in fluorescence measurements. This way, you can see how plants behave in shade and open field light conditions . Then, after that, you could add some changes in controllable factors, you can change humidity (irrigation frequency, soil type, or any other type of factor that you could be interested in to study its influence on your plants).

Dear M. Iftikhar Hussain,

I agree with Ronald Maldonado Rodriguez.

What is your hypothesis?  I understand you are looking at "ornamental plants and shrubs growing in the open field and in the shade (close to big buildings)".  If your hypothesis is that a species of ornamental plant or shrub growing in the open field will perform better than shaded individuals of the same species (or vice versa), then you can set up a sampling regime that will help address that hypothesis.  That regime would ensure, as much as possible, that every environmental variable is similar at both locations except those variables that define the differences between the two locations (light would be the obvious one, but nutrient and water availability may also be important). 

Now, assuming the chlorophyll fluorescence measurement you refer to is a PAM type measurement, then any reduction in the quantum yield of PSII photochemistry (relative to a predawn Fv/Fm measurement) will be the result of both ambient irradiance and the stressor you suspect.   As ambient irradiance effects are so important, any chlorophyll fluorescence measurements must be made in such a way that you can confidently "remove" ambient irradiance effects, leaving you with a reduction in quantum yield that is a consequence of the stressor.

However, if shading (by buildings) is the effect you hypothesise, then perhaps a better approach may be to measure effective quantum yield and irradiance over a 24 hour period at both locations and calculate electron transport rate (ETR), and non-photochemical quenching (NPQ) or the entire daytime period.  The "better" plant would have a higher ETR and less NPQ over that 24 h period, while a plant less fit may transport less electrons through PSII, and allocate more resource to NPQ, which is a non-productive suite of processes.

As Ronald Maldonado Rodriguez so eloquently stated, there are many factors to take into account before proceeding with taking measurements.  Actually, experimental design for chlorophyll fluorescence measurements is in my opinion ~80% of the hard work.  Taking the measurements themselves is relatively straightforward.

I hope this helps.

John

Dear Prof. Rodriguez and Prof. Runcie

Thank you very much for both of you for such a comprehensive reply.

Best regards

Iftikhar

Dear M. Iftikhar Hussain, Chlorophyll fluorescence and photosynthesis measurements are very important factors in plant leaf. The two parameters are very related. To measure photosynthesis, the time after morning and before midday where leaves do the process efficiently, is recommended. On the other hand chlorophyll fluorescence is measured in the night and if you want to do it in day, it is necessary you cover the leaves.   

Good Luck!

Thanks Girma.

Dear M. Iftikhar Hussain,

Together with the discussion made on the answers, you should to see that what kind of chlorophyll measurements and which type of protocol you are using, maximum quantum yield of PSII (FV/FM) or just PSII efficiency (Fv'/Fm'), NPQ or RFD. apart from that the age of the leaf also important which influence the result. I recommend to read many papers that Strasser or Kaleji written. Also you can read the following paper indicating the leaf age and light intensity dependency of fluorescence parameters:

Article Dependency of Growth, Water Use Efficiency, Chlorophyll Fluo...

Just avoid mid-day depression if it's a dark adaptation.