analysis of ammonium and nitrate in plants

Steve Houghton & Cristina Cruz ambrosia at TAKETHISOUTmail.telepac.pt
Wed Jul 15 17:13:13 EST 1998


In June I asked the group for advice about methods for analysis of nitrate
and ammonium in plant materials. I received many interesting replies, some
of which are compiled below. I would appreciate any further comments you
may have. 
I am now trying to write a note which compares the alternatives available,
which I can send to those who are interested.

Thanks

        Cristina Cruz
        Departamento de Biologia Vegetal
        Faculdade de Ciencias de Lisboa
        Campo Grande
        Bloco C-2, Piso 4
        1700 Lisboa
        Portugal

        ambrosia at mail.telepac.pt

-----------------------------------------

From: Bruce Haines <haines at dogwood.botany.uga.edu>

I offer some thoughts which may be helpful ...and maybe not

1.  NO3 and NH4 in plant material   - is this in expressed sap, or is it
dried, powdered materila that is extracted with water or what/

2.  Methods I use for traces of NO3 and NH4 in water - rain water, stream
water, soil solution are

NO3 - Strickland, JH. D.H. and T. R. Parsons (1965 I think)  A practical
handbook of seawater analysis. Fisheries research board of canada. Ottawa.

I have the 1972 edition  Bulletin 167 which describes the use of cadmium
reduction. In the 1965 edition they described the reduction with CuSO4 +
hydrazine -SO4.   I don't not have a copy of the 1965 edition in front of
me.  In the 1972 edition they say that they quit using the
hydrazine-copper approach because of mixing of air into the reduction
media with the motion of the ship.  In my lab on land, I get very
reproducable results with the hydrazine-Copper reduction.

Would this work for you?   Would your initial concentrations of nitare be
high enough that you could dillute down sample and get rid of
interferences?  I don't know.  Might be worth trying.

Could you use nitrate reductase from some garden variety plant rather than
from E. coli?   I have done nitrate reductase assays for a wide variety of
plants from herbs to pine to oak trees in a successional seequence in
South Carolina.  Leaf disks were incubated in a buffer solution containing
known amount of NO3, the buffer was vacuum infiltrated into the leaf disks
for the various plant species, the atmosphere was replaced with N2 gas,
and the stuff incubated in the dark for perhaps 30 min. What was used to
stop the reduction I don't remember. Aliquots were then assayed with
sulphanilamide and n-(1-naphthyl)-ethylenediamine dihydrochloride solution
at 543 nm.  

I wonder what happens if you took extracts from your various plant
materials and a series of nitratae standards and ran the nitrate
reduction using  disks from the same spinach leaf or tobacco leaf?  I
remember lots of nitrate reductase in the secondary successinal weeds we
sampled inthe field. I would think that spinach or tobacco would have lots
of activity

NH4  - I forget what the indophenol blue method is.  I use the method
outlines in the  1972 edition of Strickland and parson( ref given above)
which uses phenol, sodium nitroprusside, alkaline sodium citrate, and
sodoum hypochlroite.  Abs is read at 640 nm.  Is this the indophenol blue
method?

Are your concentrations high enough that you could use NO3 electrode and
an NH4 (NH3) electrode- like Orion company makes and sells?  They are
expensive.

Bruce L. Haines
Botany Department
University of Georgia
Athens, GA 30602-7271 USA
Phone 706-542-1837,FAX 542-1805
e-mail: haines at dogwood.botany.uga.edu
        haines at sparc.ecology.uga.edu
        bhaines at uga.cc.uga.edu

------------------------
From: abraham.escobar at forestry.gov.uk

I was using the microkjeldal method and I was very satisfied. 

Abraham Escobar-Gutierrez
British Forestry Commission 
Northern Research Station
Roslin, Edinburgh
Midlothian EH25 9SY
Scotland
Phone: +44 (0)131 445 2176 
Fax:   +44 (0)131 445 5124 

-----------------------------------------------------------------------

From: at6 at st-andrews.ac.uk (Alyson Tobin)
Subject: Re: analysis of ammonium and nitrate in plants

I used the methd of McCullough for ammonium determination- see Tobin AK,
Sumar N, Patel M, Moore AL, Stewart GR (1988) Development of
photorespiration during chloroplast biogenesis in wheat leaves. Journal of
Experimental Botany 39: 833-843

We used wheat leaves and found some interference in the extract. When the
extract was put through Dowex columns this removed the problem. There is
also a method for nitrate determination in this paper. It worked fine on
wheat.
I hope this helps.

Alyson Tobin

Dr A.K. Tobin
Plant Science Laboratory
School of Environmental and Evolutionary Biology
Sir Harold Mitchell Building
University of St Andrews
St Andrews
KY16 9TH
UK

-----------------------------------------------------------------------
From: MISS M M A WHITTEN <M.M.A.Whitten at swansea.ac.uk>
Organization:  University of Wales Swansea
Subject:       Re: analysis of ammonium and nitrate in plants 

    I work on insects, and have recently gone through the trials and 
tribulations of nitrate assays for extremely small blood samples.  
Originally, I wanted to try a fluorescense assay........until I found 
out about the extreme toxicity of the reagents!  The method I chose 
was the Griess colorimetric assay for nitrite (reduce nitrate to 
nitrite with bacterial nitrate reductase), and I used a kit from 
Cayman Chemical. It actually worked out cheaper (just!) to get the kit 
rather than buying in all the ingredients yourself.  
    I am no botanist, and wouldn't like to guess at all the `stuff' 
that could interfere with the assay in your samples.  I got 
interference with the Griess reagent from blood proteins, but by using 
ultrafiltration (30 kDa cut-off) the results were much better. Some 
people use zinc sulphate for deproteinizing (do this right at the end 
and then centrifuge before adding Griess reagent).
    The assay is not particularly sensitive; down to 2 micromolar is 
about the limit.  
    The technical support team at Cayman Chemical are quite helpful.  
Their website is:  http://caymanchem.com/tech.shtml.

    Hope this helps.
    
    Sorry I have no idea about assays for ammonium.

    Good luck!
            Regards,     Miranda Whitten
                         <bawhitte at swan.ac.uk>

-----------------------------------------------------------------------

From: Curtis V Givan <cgivan at christa.unh.edu>
Subject: Nitrate and ammonium assays

Dear Dr. Cruz,

I hope this information may be helpful.

For assaying nitrate, and as long as there are no problems with inhibitors
that will inhibit nitrate reductase, I can recommend the system worked out
by Campbell using corn nitrate reductase.  There is a commercial company
selling kits for this method, address:  http://www.nitrate.com (the
Nitrate Elimination Company).

For ammonia, my lab has used the phenol hypochlorite method.  It is
said to be specific for ammonia but there is interference from some
buffers such as HEPES and tricine.
Ref. McCullough, H. 1967.  The determination of ammonia in whole blood by
a direct colorimetric method.  Clin. Chim. Acta 17, 297-304.

There may be more sensitive methods available if you are needing to assay
extremely low amounts, but these are methods that don't require very
specialized equipment, and they are reliable.

Sincerely,

Curtis Givan
Date: Fri, 05 Jun 1998 08:17:06 +0200
-----------------------------------------------------------------------

From: Chris <chris at laracroft.demon.co.uk>
Subject: Re: analysis of ammonium and nitrate in plants

Hi Cristina, we use several different methods but theyre really meant
for water samples. I do have experience of using the kjeldahl method
for solids, which we have used to determine ammonia plus organic
nitrogen. You need to be careful when using this method as you'll
find any nitrate present will oxidise the ammonical nitrogen, giving
erratic results. We get round this by using a copper catalyst with
ethanol added to remove nitrate as ethyl-nitrate.

We also use a persulphate digestion system to produce total N results, it
has been applied to quite dirty samples successfully. Its not
really that time consuming as we use an autoclave to perform these
digestions, and then analyse for nitrate by reducing to nitrite (Cadmium
reduction) and using a di-azo reaction to characterise the
NO2 produced.

High (as in >2 ppm N) ammonia is done by steam distillation and
titration using a buchi.

---------------------------------
From: Donald MacKerron <D.MacKerron at scri.sari.ac.uk>
Organization: Scottish Crop Research Institute
Subject: Re: analysis of ammonium and nitrate in plants

Do you need to keep ammonium and nitrate separate?
If you can accept total N then you might be interested in either NIR or
NIT, Near Infrared Reflectance and Transmission.
I haven't used NIT which is the more modern technique.
We have used NIR extensively on dried material.  I think that, if you
have the appropriate hardware, you can use fresh material.

You will find some accounts of the techniques in the following
references:

MacKerron, D.K.L., Young, M.W. and Davies, H.V., 1993.  A method to
optimize N-application in relation to soil supply of N and yield in
potato.  Plant and Soil  155, 139-144.

Young, M.W., Davies, H.V. & MacKerron, D.K.L., 1993.
Comparison of techniques for nitrogen analysis in potato crops.  In:
Optimization of Plant Nutrition.
Proceedings VIIIth International Colloquium for the Optimization of
Plant Nutrition.  M.A.C. Fragoso & M.L. van Beusichem (eds.).  pp 7-11.

Young, M.W., MacKerron, D.K.L., and Davies, H.V.,  1997.
Calibration of near infrared reflectance spectroscopy to estimate
nitrogen concentration in
potato tissues.  Potato Research 40, 215 - 220.

 MacKerron, D.K.L., Young, M.W. and Davies, H.V., 1997.
Factors influencing the calibration of near infrared reflectometry (NIR)
applied to the assessment
of total nitrogen in potato tissues:  I.  Particle size, milling speed,
and leaf senescence.  Journal of Near Infrared Spectroscopy 3, 155 -
166.

Young, M.W., MacKerron, D.K.L., and Davies, H.V.,  1997.
Factors influencing the calibration of near infrared reflectometry (NIR)
applied to the
assessment of total nitrogen in potato tissues.  2.  Operator, Moisture
and Maturity Class.  Journal of Near Infrared Spectroscopy 3, 167 - 174.

Best wishes,
Donald MacKerron
Tel: +44 (0)1382 562731
Fax: +44 (0)1382 562426
E-mail:  D.MacKerron at scri.sari.ac.uk

------------------------

From: "David Guillemant" <dguillemant at nordnet.fr>
Subject: Re: analysis of ammonium and nitrate in plants

Maybe you ll find some information in a new french book (INRA ed.) about
assimilation of N in plants. I don't know excatly but I beleive it has been
written i english.
english of the book is surely better than "my" english.


I don't know the web adress of INRA but it will be easy to find it.

sincerly, 

DAVID

----------------------------------------------------------------------
From: Robert Perrin <Robert.Perrin at dijon.inra.fr>
Subject: Re: analysis of ammonium and nitrate in plants
Organization: INRA

Voici donc les coordonnées du web INRA 

Following you will find the Web adress of INRA where you will find all
the informations you need including a list of researcher involved in N
studies. May I recommend you to look at COMPACT item !

Good luck

BOB

http://www.inra.fr/

-- 
Steve Houghton & Cristina Cruz
ambrosia at mail.telepac.pt



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