Non-aqueous Fractionation (NAF) for Metabolite Analysis in Subcellular Compartments of Arabidopsis Leaf Tissues.

The accurate determination of metabolite distribution in subcellular compartments is still challenging in plant science. Various methodologies, such as fluorescence resonance energy transfer-based technology, nuclear magnetic resonance spectroscopy and protoplast fractionation allow the study of metabolite compartmentation. However, large changes in metabolite levels occur during such procedures. Therefore, the non-aqueous fractionation (NAF) technique is currently the best method for the study of in-vivo metabolite distribution. Our protocol presents a detailed workflow including the NAF procedure and quantification of compartment-specific markers for three subcellular compartments: ADP glucose pyrophosphorylase (AGPase) as plastidic marker, phosphoenolpyruvate carboxylase (PEPC) as cytosolic marker, and nitrate and acid invertase as vacuolar markers.

amount of tissue material (Fürtauer et al., 2016). This benchtop NAF method can be used as an alternative to the conventional NAF procedure. However, we still believe that the conventional NAF procedure is the method of choice for the determination of in vivo metabolite distribution among cellular compartments as (i) the plastidic and vacuolar compartments are more clearly separated on the density gradient used, and (ii) the higher amount of starting material leads to more material in each obtained fraction, giving the advantage to increase the number of sub-aliquots per fraction (thus increasing the range of measurements which can be done) and also to apply less sensitive quantification methods than mass spectrometry-based methods. A good example of the use of the conventional NAF procedure is a study performed by Arrivault et al. (2014) in which the distribution of about 1,000 proteins and 70 metabolites, including 22 phosphorylated intermediates in Arabidopsis thaliana rosette leaves were analyzed, using the conventional NAF combined with MS-based approaches.
The protocol presented here is based on the method described by Krueger et al. (2014) with minor modifications. In our method, we provide a complete workflow for the NAF procedure in Arabidopsis thaliana and detailed information for the analysis of compartment-specific marker enzymes and metabolites which can be assigned to three subcellular compartments: ADP glucose pyrophosphorylase (AGPase) as plastidic marker, phosphoenolpyruvate carboxylase (PEPC) as cytosolic marker and nitrate and acid invertase as vacuolar markers.

Materials and Reagents
A. NAF     Alternatively, the grinding of the leaf material can be performed by using a pre-cooled mortar and pestle. Make sure that the leaf material is kept frozen during the whole procedure.  sonication is successful. This is a critical step in the fractionation, since insufficient sonication can produce aggregates that do not pass through nylon mesh.

system.
c. Set the peristaltic pump at a flow rate of 1.15 ml min -1 and the following program: Step  Figure 3B). Re-start the program.

Note: We always use the Ultracentrifuge Beckman Coulter Optima TM L80 XP and Swinging bucket rotor Beckman Coulter SW32Ti. Set the acceleration/deceleration on three.
10. Place the gradient tubes in a rack and mark eight fractions. Figure 4 illustrates how we usually separate the fractions based on the color.

and determination of the compartment-specific markers.
14. Transfer 6 x 1 ml from each suspension into 1.5 ml screw caps tubes. The pellet can easily reaggregate at this step. Keep mixing by hand the solution while transferring into the tubes in order to obtain homogeneous aliquots from the same fraction.
Note: Use a cut 1000 µl pipette tip to collect sub-aliquots from each fraction. As adding 7 ml generally does not allow to obtain seven aliquots of 1 ml, we generally take 0.8 ml for the seventh aliquot and clearly label the corresponding tube.

Nitrate (vacuolar marker)
Nitrate content is determined following Cross et al. (2006). Nitrate can be used as a vacuolar marker in addition or alternatively to Acid Invertase.  2. For the values of either markers and metabolites of interest in the input file, we recommend using percentage, although absolute measurements can also be used (note that in this case values must be ≥ 0).
3. For more details on how to use BestFit, we suggest reading the documentation pdf file within the BestFit Folder (http://www.csbdb.de/csbdb/bestfit/bestfit.html). Alternatively, you can also find details regarding data input and use of BestFit in Krueger et al. (2014). 4. BestFit outputs the subcellular-distribution of the metabolites of interest in percentage. In order to calculate which amounts this represents, metabolites are also measured in ground material not processed through NAF (see Note in A1). Various recovery calculations can be done by using the following information: (i) material fresh weight used for NAF (determined in step A4), (ii) corresponding dry weight (determined in step A6), (iii) how much material was applied to the gradient (determined in step C18 by weighing the residual material in the filter), (iv) F0 obtained step C6. We recommend always recording these data and collecting F0 for each NAF. Prepare only prior the extraction combining as the following: