This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany&#8217;s Excellence Strategy - EXC-2070 &#8211; 390732324 (PhenoRob), the Research Training Group GRK2064 and individual research grants SCHA1274/4-1 and SCHA1274/5-1 to G.S.. We also thank Li Schl&#252;ter and Brigitte Ueberbach for technical assistance.

The authors have nothing to disclose.

Here we present a versatile and sensitive method to quantify InsPs including PP-InsPs in plant extracts and provide practical tips on how to get this method established. Even though the protocol is generally robust, suboptimal runs and analyses can occur. In most cases, those runs can be identified by a strong reduction or even complete loss of highly phosphorylated InsPs, especially the PP-InsP species InsP7 and InsP8. Possible reasons can be microbial contaminations of the plant material and insufficient deactivation of endogenous plant PP-InsP hydrolases during extraction due to insufficient grinding and thawing of plant material that will not be in immediate contact with extraction buffer. Further reasons include inaccurate pH adjustment by insufficient or excess addition of neutralization buffer, or simply insufficient sample material. The latter can make it difficult to detect PP-InsPs, since those are often present in very low amounts in the cells. An excess of sample material or inefficient drying during step 3.5 may cause dilution of the perchloric acid, therefore also leading to insufficient enzyme deactivation and a specific loss of InsP6 and PP-InsPs. The amount of plant material, as well as radiolabel used in this protocol were optimized based on costs and performance, and is therefore close to the lowest amount that is still sufficient for providing optimal results. In addition, the column resin will gradually loose its resolution&#160;capacity. The first sign of this process is (for reasons not entirely clear to the authors) a specific loss of higher phosphorylated InsP species like the PP-InsPs in the HPLC spectrum. With further aging, even InsP6 will not be resolved properly by the column (Figure 1D). Therefore, the use of an adequate column, as well as meticulous handling of the sample and proper maintenance of the HPLC components is crucial for ensuring accurate results.
When comparing samples and runs, especially when generated with different equipment (e.g., HPLC systems and columns) or on different days, it is crucial to normalize the samples to each other (as described in step 7.3) and to analyze them in the same way. Only through normalization it is possible and accurate to show multiple samples in the same graph (Figure 3). For quantification of individual InsPs relative to total InsPs, or to another specific InsP species, it is not necessary to normalize, as long as only relative values and not absolute values are shown. Ideally, both the InsP profiles and the quantifications are shown. However, in some cases it is not possible to adequately show two or more runs in the same graph. Different retention times or different levels of background activity can make it difficult to compare unquantified SAX-HPLC profiles alone. The same is the true when many samples need to be compared. In such cases, a further evaluation using an additional software (e.g., Origin) for individual peak quantification is necessary.
The authors are aware that the protocol described here can be optimized and needs to be adapted to each individual research question. Although being optimized for Arabidopsis extracts7,17 in this protocol, this method is versatile and can help determining InsP profiles of other plant species as well. Here we exemplify this possibility by presenting for the first time a InsP profile for L. japonicus, which required no modifications of the labeling conditions, InsP extraction or SAX-HPLC run (Figure 2). Notably, while overall similar, differences are observed between L. japonicus and Arabidopsis InsP profiles. For instance, in L. japonicus InsP5 [4-OH] or its enantiomeric form InsP5 [6-OH] are more abundant than InsP5 [1-OH] or its enantiomeric form InsP5 [3-OH] in comparison to Arabidopsis, where InsP5 [1-OH] or its enantiomeric form InsP5 [3-OH] are the dominant InsP5 species. Likewise, we anticipate that alterations in the media composition, [3H]-myo-inositol concentration, plant age, environmental conditions (e.g., light and temperature), addition of chemical compounds or analyses of plant-microbial interactions among other factors, might need to be tested and adapted.
One important drawback of this method that needs to be considered is that the labeling is done in a (sterile) liquid culture, which does not represent a physiological environment for most land plants. In addition, due to the high costs of [&#179;H]-myo-inositol, the volume of the labeling solution and the size of the culture vessel is generally limited, which also restricts the size of the plants that can be used. Cultivation in liquid culture can be avoided by directly infiltrating for instance leaves of soil-grown plants with [&#179;H]-myo-inositol and subsequently following the protocol described here, as previously reported10.
There are several drawbacks of this protocol in comparison to alternative methods, such as TiO2 pull-down followed by PAGE or mass spectrometry based techniques. Due to the [3H]-myo-inositol labeling, only InsP species that directly originate from radiolabeled myo-inositol will be detected in the end. The method described here is blind to other Ins isomers such as scyllo-inositol and other isomers some of which have been identified in certain plants44. Furthermore, myo-InsPs derived from other pathways will be excluded, including those synthesized by de novo synthesis of myo-inositol and myo-inositol-3-phosphate via isomerization of glucose-6-phosphate, catalyzed by myo-inositol-3-phosphate synthase (MIPS) proteins45. Although [32P] or [33P]-ortho-phosphate can be used as alternative labels, their use poses a major disadvantage, since every phosphate-containing molecule, including the abundant nucleotides and its derivatives, will be labeled. Those molecules can also be extracted with this protocol and bind to the SAX column, which will result in a high level of background activity that will interfere with the identification of individual InsP peaks5. In addition, quantification of [32P]- or [33P] -labeled InsPs and PP-InsPs can be strongly influenced by phosphate and pyrophosphate moiety turnover and might not report a mass readout for inositol species.
On the other hand, [3H]-myo-inositol specifically labels myo-inositol-containing molecules. InsPs, inositol-containing lipids, such as phosphoinositides, and galactinol are in this case labeled. However, only InsPs will be analyzed with this protocol, since lipids are insoluble in the extraction buffer and galactinol does not bind to the SAX&#160;column.
So far, the differences from a plant InsP profile generated by [3H]-myo-inositol labeling compared with one determined by TiO2 pulldown/PAGE remains unknown, since such comparisons have not been performed in plants. A recent study in animal cells addressed this question46. In that work, a pool of InsP6 that is invisible by [3H]-myo-inositol labeling, which should thereby be directly derived from glucose-6-phosphate, was identified by comparing SAX-HPLC profiles with PAGE gels of mammalian cell lines. 24 h of phosphate starvation resulted in a 150% increase of InsP6 when quantifying PAGE gels of InsPs purified using TiO2 pulldown. SAX-HPLC analyses of [3H]-myo-inositol-labeled cells that were treated identically only showed an increase by 15% of [3H]-InsP6. As previously mentioned, InsPs lower than InsP5 are undetectable with PAGE analysis in most cases. Radiolabeling followed by SAX-HPLC appears to be the method of choice, as long as mass spectrometric protocols are not optimized to detect this group of highly negatively charged molecules.
Another remaining challenge is to distinguish enantiomers in SAX-HPLC analyses (or in any other method for InsP analysis)10,17. This challenge can be tackled by the addition of chiral selectors, i.e., enantiopure compounds like L-arginine amide that interact with the respective enantiomeric molecules to form diastereomeric complexes that can be separated10. To our knowledge, this approach has only been implemented to discriminate the enantiomeric InsP5 isomers InsP5&#160;[1-OH] and InsP5 [3-OH] by NMR analyses10. Discrimination of other enantiomeric pairs or successful discrimination of enantiomers by chiral SAX-HPLC analysis or chiral PAGE-based methods have not yet been reported and should be further developed. Considering the conserved synthesis and the conserved regulation of PP-InsPs by phosphorous availability, we envision that especially non-radioactive methods such as PAGE or MS-based methods, together with nutrient analyses, will help ground truthing efforts to calibrate remote sensing data designed to diagnose nutrient deficiencies in crops17,18,24,25. However, the method presented here can currently still be considered the gold standard for InsP analyses and will be instrumental to discover new functions of these intriguing messengers in plants.

Almost four decades ago, inositol phosphates (InsPs) emerged as signaling molecules, after Ins(1,4,5)P3 (InsP3) was identified as a second messenger that activates the receptor-mediated release of Ca2+ in animal cells1,2. To date, no InsP3 receptor (IP3-R) has been identified in plants, which questions a direct signaling role for InsP3 in plant cells3. Regardless, InsP3 serves as a precursor for other InsPs involved in several plant developmental processes, including the regulation of specific signaling pathways3,4,5,6,7,8. For instance, InsP3 can be further phosphorylated to InsP6, also known as &#8220;phytic acid&#8221;, which represents a major source of phosphate, myo-inositol and cations, and was shown to play key roles in plant defense against pathogens, mRNA export and phosphate homeostasis5,9,10,11,12.
Inositol pyrophosphates (PP-InsPs) are a class of InsPs that contain at least one high-energy di-phospho bond, initially identified in animal cells, amoeba and yeast, where they play critical roles in various cellular processes13,14,15. Despite seminal work on PP-InsPs in plants16,17,18,19,20,21,22,23,24,25,26, the biological functions and isomer identity of these molecules still remain largely enigmatic. In the model plant Arabidopsis thaliana, cellular InsP8 was proposed to regulate defenses against insect herbivores and necrotrophic fungi via coincidence-detection of InsP8 and active jasmonate by the ASK1-COI1-JAZ receptor complex17. Furthermore, roles of InsP8 and other PP-InsPs in energy homeostasis and nutrient sensing, as well as phosphate homeostasis have been proposed17,23,24,25,26.
Regardless of the biological system employed, one major methodological challenge when studying InsPs has been the reliable detection and precise quantification of these molecules. Mass spectrometry-based methods have been used to detect InsPs, including PP-InsPs, from cell extracts.&#160;However,&#160;those studies failed to differentiate distinct isomers26,27. Another approach to analyze InsPs employs pull-down of InsPs from cell lysates using TiO2 beads, followed by polyacrylamide gel electrophoresis (PAGE) of the eluted InsPs. The InsPs can then be stained by either toluidine blue or DAPI24,28,29. However, it is so far not possible to reliably detect InsPs lower than InsP5 from plant extracts using this method. Recently, a method using [13C]-myo-inositol for nuclear magnetic resonance (NMR) analysis of InsPs was published as an alternative to strong anion exchange high-performance liquid chromatography (SAX-HPLC)30. This technique has been reported to achieve a similar sensitivity compared to SAX-HPLC and to allow the detection of 5-InsP7, as well as the discrimination of different non-enantiomeric InsP5 isomers from cell extracts. However, the implementation of the NMR-based method&#160;requires chemically synthesized and commercially not available [13C]-myo-inositol. Therefore, the method employed in most cases is radiolabeling samples with [3H]-myo-inositol, followed by SAX-HPLC31,32,33. This technique is based on the uptake of radioactive myo-inositol into the plant and its conversion into different InsPs by the combined activity of dedicated cellular kinases and phosphatases.
The [3H]-labeled InsPs are then acid-extracted and fractionated using SAX-HPLC. Because of their negative charge, the InsPs strongly interact with the positively charged stationary phase of the SAX-HPLC column and can be eluted with a buffer gradient containing increasing phosphate concentrations to outcompete InsPs from the column. Elution times thus depend on charge and geometry of the InsP species to be separated. In the absence of chiral columns, only non-enantiomeric isomers can by separated by this protocol. However, radiolabeled standards can be used to assign the isomeric nature of a specific InsP peak. Multiple efforts in the past by various laboratories to generate labeled and unlabeled standards with (bio)chemical methods or to purify them from various cells and organisms have helped assigning peaks to certain InsP species, and also to narrow down the isomeric identity of individual InsP species5,7,21,34,35,36,37,38,39,40,41,42,43. Also, the recent elucidation of enzymatic pathways leading to the formation of PP-InsPs in plants, as well as the discovery of a bacterial type III effector with a specific 1-phytase activity, provide information on how to generate useful standards for these analyses10,17,18,22,23.
The resulting fractions can be measured in a liquid scintillation counter due to the &#946;-decay of tritium (3H). With increasing labeling time, a steady-state isotopic equilibrium is reached, after which the obtained InsP profiles should represent the InsP status of the plant31. The major advantage of this protocol in comparison to other available techniques is the high sensitivity achieved by the use of the direct precursor for InsPs and the measurement of a radioactive signal.
SAX-HPLC of samples extracted from [3H]-myo-inositol-labeled plants or other organisms is commonly used for the detection and quantification of InsPs ranging from&#160;lower InsP species to&#160;PP-InsPs, representing a valuable tool to better understand the metabolism, function and modes of action of InsPs. So far, this method is also the most appropriate choice for researchers with special interest in lower InsP species. While the basics of this procedure, on which the protocol described here builds on, have been previously described7,21,31,34, a detailed protocol tailored to the analysis of plant-derived InsPs and especially of PP-InsPs is still missing. Previous publications reported difficulties to reliably detect the low abundant PP-InsPs, especially InsP8, due to one or more of the following factors: relatively low amounts of plant material, [3H]-myo-inositol with low specific activity (&#62; 20 Ci/mmol), usage of&#160;extraction buffers that are either not based on perchloric acid or are less concentrated than 1 M, different neutralizing buffers, as well as sub-optimal gradients or detection of [3H] with an on-line detector. In comparison to those studies, the protocol presented here is designed for the reliable detection of PP-InsPs7,21,34.
Here we present a detailed workflow, starting from the setup of the equipment to plant cultivation and labeling, InsP extraction and the SAX-HPLC run itself. Although the method was optimized to the model plant A. thaliana, it can be easily modified to study other plant species, as shown here with the first reported InsP profile of the model legume Lotus japonicus. Although the use of a different plant species might require some optimization, we envisage that those will be minor, making this protocol a good starting point for further research in plant InsPs. In order to facilitate possible optimizations, we indicate every step within the protocol in which modifications are possible, as well as all critical steps that may be challenging when establishing the method for the first time. Additionally, we report how data obtained by this method can be used for the quantification of specific InsPs and how different samples can be analyzed and compared.

<table>
	<tbody>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf AG</td>
			<td>model: 5430 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Diammonium hydrogen phosphate, &#8805;97 %</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>0268.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediamine tetraacetic acid disodium salt dihydrate, &#8805;99 %, p.a., ACS</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>8043.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 12-well clear flat bottom TC-treated multiwell cell culture plate, with lid, individually wrapped, sterile</td>
			<td>Corning Inc.</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>Fraction collector</td>
			<td>LAMBDA Instruments GmbH</td>
			<td>model: OMNICOLL single channel collector</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth incubator</td>
			<td>poly klima GmbH</td>
			<td>model: PK 520-LED</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC pumps</td>
			<td>Kontron Instruments</td>
			<td>model: 420</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC syringe for Rheodyne valves, 1 mL</td>
			<td>Hamilton Company</td>
			<td>81365</td>
			<td></td>
		</tr>
		<tr>
			<td>Injector for HPLC</td>
			<td>Supelco</td>
			<td>model: Rheodyne 9725</td>
			<td></td>
		</tr>
		<tr>
			<td>Inositol, myo-[1,2-3H(N)]</td>
			<td>American Radiolabeled Chemicals Inc.</td>
			<td>ART 0261</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>University, Chemistry Department</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid scintillation counter</td>
			<td>PerkinElmer Inc</td>
			<td>model: TRI-CARB 2900TR</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro pestle</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>CXH7.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixed cellulose Eester filter, ME range (ME 24), plain, 0.2 &#181;m pore size, 47 mm circle</td>
			<td>GE Healthcare Life Sciences</td>
			<td>10401770</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixer for HPLC</td>
			<td>Kontron Instruments</td>
			<td>model: M 800</td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige &#38; Skoog medium, salt mixture</td>
			<td>Duchefa Biochemie</td>
			<td>M0221</td>
			<td></td>
		</tr>
		<tr>
			<td>OriginPro software</td>
			<td>OriginLab Corp.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orthophosphoric acid, &#8805;85 %, p.a., ISO</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>6366.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Partisphere 5 &#181;m SAX cartridge column, 125 x 4.6 mm</td>
			<td>Hichrom Limited</td>
			<td>4621-0505</td>
			<td></td>
		</tr>
		<tr>
			<td>Perchloric acid, 70 %, 99.999 % trace metals basis</td>
			<td>Sigma-Aldrich</td>
			<td>311421</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, square, PS, clear, 120/120/17 mm, sterile</td>
			<td>Greiner Bio One International GmbH</td>
			<td>688161</td>
			<td></td>
		</tr>
		<tr>
			<td>pH-indicator paper pH 5.5 - 9.0, Neutralit</td>
			<td>Merck KGaA</td>
			<td>109564</td>
			<td></td>
		</tr>
		<tr>
			<td>Phytagel</td>
			<td>Sigma-Aldrich</td>
			<td>P8169</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium carbonate</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>P743.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock tubes, 1.5 mL</td>
			<td>Eppendorf AG</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample loop for 9725 injectors, volume 2 mL, PEEK</td>
			<td>Supelco</td>
			<td>57648</td>
			<td></td>
		</tr>
		<tr>
			<td>SNAPTWIST scintillation vial, 6.5 mL</td>
			<td>Simport Scientific Inc.</td>
			<td>S207-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile bench</td>
			<td>LaboGene</td>
			<td>model: ScanLaf MARS 900</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose, &#8805;99,5 %, p.a.</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>4621.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultima-Flo AP liquid scintillation cocktail</td>
			<td>PerkinElmer Inc</td>
			<td>6013599</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-pure deionized water</td>
			<td>Milli-Q</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wrenchless WVS End Fitting Kit</td>
			<td>Hichrom Limited</td>
			<td>4631-1001</td>
			<td></td>
		</tr>
	</tbody>
</table>

extraction and quantification of soluble, radiolabeled inositol polyphosphates from different plant species using sax-hplc

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant physiology. Due to their negative charge, low abundance and susceptibility to hydrolytic activities, the detection and quantification of these molecules is challenging. This is particularly the case for highly phosphorylated forms containing &#8216;high-energy&#8217; diphospho bonds, also termed inositol pyrophosphates (PP-InsPs). Due to its high sensitivity, strong anion exchange high-performance liquid chromatography (SAX-HPLC) of plants labeled with [3H]-myo-inositol is currently the method of choice to analyze these molecules. By using [3H]-myo-inositol to radiolabel plant seedlings, various InsP species including several non-enantiomeric isomers can be detected and discriminated with high sensitivity. Here, the setup of a suitable SAX-HPLC system is described, as well as the complete workflow from plant cultivation, radiolabeling and InsP extraction to the SAX-HPLC run and subsequent data analysis. The protocol presented here allows the discrimination and quantification of various InsP species, including several non-enantiomeric isomers and of the PP-InsPs, InsP7 and InsP8, and can be easily adapted to other plant species. As examples, SAX-HPLC analyses of Arabidopsis thaliana and Lotus japonicus seedlings are performed and complete InsP profiles are presented and discussed. The method described here represents a promising tool to better understand the biological roles of InsPs in plants.

The results shown here aim to illustrate possible outcomes obtained according to variations at technical and biological levels. The first is exemplified by analyses using new versus aged columns (Figure 1) and fresh versus stored samples (Figure 3), and the second by evaluating extracts from two different plant systems, A. thaliana (Figure 1, Figure 3) and L. japonicus (Figure 2).
An optimal SAX-HPLC run is depicted on Figure 1A&#8210;C, which shows a complete inositol polyphosphate spectrum obtained from A. thaliana extracts after scintillation counting. Note that peaks are nicely separated and can be assigned to different isomers (or enantiomer-pairs) based on chromatographic mobilities described earlier5,7.
Figure 2 shows the representative result of a SAX-HPLC analysis of L. japonicus seedlings that were grown and labeled under the same conditions as the Arabidopsis seedlings. While presumably all InsP species and peaks that are known from Arabidopsis can be seen, there are substantial differences regarding the relative (e.g., ratios between isomers) amount of specific InsP isomers, when comparing the profiles of both species. For instance, the Lotus extracts showed increased InsP3c, InsP4b, InsP5b and reduced InsP3a, InsP4a, InsP5a and InsP5c compared to Arabidopsis which leaves room for further investigations. Figure 2D illustrates the different ratios between InsP isomers between Arabidopsis and Lotus.
Figure 3 shows two InsP profiles of a sample that was split after the extraction. The first half was immediately analyzed and the second half one day later, after storage at -80 &#176;C. Note that only minor&#160; differences are observed between the different samples (i.e., black and red lines on Figure 3A&#8210;C, and Figure 3D). This illustrates that one freeze-thaw cycle does not harm the sample and that the method itself generates reproducible results.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61495/61495fig01.jpg" /> 
Figure 1: Typical InsP profile of a successful and of an unsuccessful SAX-HPLC analysis performed with this protocol.&#160;(A&#8210;C) SAX-HPLC profile of 17-day-old wild-type (Col-0) Arabidopsis seedlings radiolabeled with [3H]-myo-inositol. Global InsP extraction and SAX-HPLC run were performed on the same day. (A) Full spectra; (B, C) Zoom-ins of the profile shown in A. All visible peaks are highlighted and assigned to the corresponding InsP species. Based on published chromatographic mobilities5,7, InsP4a likely represents Ins(1,4,5,6)P4 or Ins(3,4,5,6)P4, InsP5a represents InsP5 [2-OH], InsP5b represents InsP5 [4-OH] or its enantiomeric form InsP5 [6-OH], and InsP5c represents InsP5 [1-OH] or its enantiomeric form InsP5 [3-OH]. The isomeric natures of InsP3a-c, InsP4b, InsP7, and InsP8 are still unknown. Panel (D) shows a SAX-HPLC profile of identically grown plants but using an aged column (&#62;40 runs). A clear reduction of InsP6 compared to other InsP species and the absence of PP-InsPs is visible. <a href="https://www.jove.com/files/ftp_upload/61495/61495fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61495/61495fig2v2.jpg" /> 
Figure 2: Representative InsP profile of L. japonicus plants.&#160;SAX-HPLC profile (A&#8210;C) of 17-day-old wild-type (Gifu) L. japonicus seedlings radiolabeled with [3H]-myo-inositol. (A) Full spectra; (B, C) Zoom-ins of the profile shown in A. All visible peaks are highlighted and assigned to the corresponding InsP species. Based on published chromatographic mobilities5,7, InsP4a likely represents Ins(1,4,5,6)P4 or Ins(3,4,5,6)P4, InsP5b likely represents InsP5 [4-OH] or its enantiomeric form InsP5 [6-OH], and InsP5c likely represents InsP5 [1-OH] or its enantiomeric form InsP5 [3-OH]. The isomeric natures of InsP3a-c, InsP4b, InsP7, and InsP8 are unknown. (D) Comparison between the individual InsP species (in % of total activity from elution 25&#8210;96) of A. thaliana (data from Figure 1A&#8210;C) and L. japonicus (data from Figure 2A&#8211;C). <a href="https://www.jove.com/files/ftp_upload/61495/61495fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61495/61495fig03.jpg" /> 
Figure 3: InsP profiles of a split sample illustrating the reproducibility of SAX-HPLC analyses.&#160;(A&#8210;C) SAX-HPLC profiles of 17-day-old wild-type (Col-0) Arabidopsis seedlings radiolabeled with [3H]-myo-inositol. Prior the run, the sample was split and one half run immediately and the other half one day later after storage at -80 &#176;C. (A) Full spectra; (B, C) Zoom-ins of the profile shown in A. All visible peaks are highlighted and assigned to the corresponding InsP species. Based on published chromatographic mobilities5,7, InsP4a likely represents Ins(1,4,5,6)P4 or Ins(3,4,5,6)P4, InsP5a represents InsP5 [2-OH], InsP5a represents InsP5 [2-OH], InsP5b represents InsP5 [4-OH] or its enantiomeric form InsP5 [6-OH], and InsP5c represents InsP5 [1-OH] or its enantiomeric form InsP5 [3-OH]. The isomeric natures of InsP3a-c, InsP4b, InsP7, and InsP8 are still unknown. Panel D shows the quantification of InsP6 and the PP-InsPs InsP7 and InsP8 of both runs. The values represent the amount (in %) of the respective InsP species relative to all InsP (total activity from elution 25&#8211;96). <a href="https://www.jove.com/files/ftp_upload/61495/61495fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure 1: Software settings for liquid scintillation counting using a light scintillation counter. Screenshots showing the software version, as well as settings used for scintillation counting of [3H] samples performed with this protocol are depicted. <a href="https://cloudflare.jove.com/files/ftp_upload/61495/61495supfig01.jpg" target="_blank">Please click here to download this figure.</a>
Supplemental Figure 2: Representative example of data normalization. A screenshot of a worksheet shows all steps and formulas used to normalize SAX-HPLC runs to each other. For simplification, only fractions 25&#8211;35 of samples are shown. <a href="https://cloudflare.jove.com/files/ftp_upload/61495/61495supfig02.jpg" target="_blank">Please click here to download this figure.</a>
Supplemental Figure 3: Peak determination, background subtraction and integration using analysis software. (A) Data from SAX-HPLC analysis is loaded into the software (minutes 28&#8211;96) and the peak analyzer tool is selected. (B&#8210;E) The baseline is defined manually by setting points between individual peaks and the background is subtracted. (F) Peaks are determined manually based on appearance and published chromatographic mobilities5,7. (G) Peak ranges are defined manually by cpm values. (H) Peaks are integrated and calculated as % of all peaks. <a href="https://cloudflare.jove.com/files/ftp_upload/61495/61495supfig03.jpg" target="_blank">Please click here to download this figure.</a>

Watch this Scientific Journal Video about Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC at JoVE.com

Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC

Here we describe strong anion exchange high-performance liquid chromatography of [3H]-myo-inositol-labeled seedlings which is a highly sensitive method to detect and quantify inositol polyphosphates in plants.

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant ...

extraction-quantification-soluble-radiolabeled-inositol

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Biochemistry

6.9K Views.  University of Bonn. Here we describe strong anion exchange high-performance liquid chromatography of [3H]-myo-inositol-labeled seedlings which is a highly sensitive method to detect and quantify inositol polyphosphates in plants.

Video: Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a key pathological feature of HD is the aberrant accumulation of mutant HTT (mHTT) protein into high molecular weight complexes and intracellular inclusion bodies composed of fragments and other proteins. Conventional methods to measure and understand the contributions of various forms of mHTT-containing aggregates include fluorescence microscopy, western blot analysis, and filter trap assays. 
However, most of these methods are conformation specific, and therefore may not resolve the full state of mHTT protein flux due to the complex nature of aggregate solubility and resolution.
For the identification of aggregated mHTT and various modified forms and complexes, separation and solubilization of the cellular aggregates and fragments is mandatory. Here we describe a method to isolate and visualize soluble mHTT, monomers, oligomers, fragments, and an insoluble high molecular weight (HMW) accumulated mHTT species. HMW mHTT tracks with disease progression, corresponds with mouse behavior readouts, and has been beneficially modulated by certain therapeutic interventions1. This approach can be used with mouse brain, peripheral tissues, and cell culture but may be adapted to other model systems or disease contexts.

A method is described for fractionation of insoluble and soluble mutant huntingtin species from mouse brain and cell culture. The method described is useful for characterization and quantification of huntingtin protein flux and aids in analyzing protein homeostasis in disease pathogenesis and in the presence of perturbations

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a chemical defense because it has antimicrobial activity and is considered a natural insecticide. Caffeine can also produce negative allelopathic effects that prevent the growth of surrounding plants. In addition, people around the world consume caffeine for its analgesic and stimulatory effects. Due to interest in the technological applications of caffeine, research on the biosynthetic pathway of this compound has grown. These studies have primarily focused on understanding the biochemical and molecular mechanisms that regulate the biosynthesis of caffeine. In vitro tissue culture has become a useful system for studying this biosynthetic pathway. This article will describe a step-by-step protocol for the quantification of caffeine and for measuring the transcript levels of the gene (CCS1) encoding caffeine synthase (CS) in cell suspensions of C. arabica L. as well as its activity.

This protocol describes an efficient methodology for the extraction and quantification of caffeine in cell suspensions of C. arabica L. and an experimental process for evaluating the enzymatic activity of caffeine synthase with the expression level of the gene that encodes this enzyme.

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique. &#160;

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid (TGA)

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls and is an aromatic heteropolymer primarily composed of three monolignols with significant industrial importance. Lignin plays an important role in plant growth and development, protects&#160;from biotic and abiotic stresses, and in the quality of animal fodder, the wood, and industrial lignin products. Accurate estimation of lignin content is essential for both fundamental understanding of the lignin biosynthesis and industrial applications of biomass. The thioglycolic acid (TGA) method is a highly reliable method of estimating the total lignin content in the plant biomass. This method estimates the lignin content by forming thioethers with the benzyl alcohol groups of lignin, which are soluble in alkaline conditions and insoluble in acidic conditions. The total lignin content is estimated using a standard curve generated from commercial bamboo lignin.

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid TGA

Here, we present a modified TGA method for estimation of lignin content in herbaceous plant biomass. This method estimates the lignin content by forming specific thioether bonds with lignin and presents an advantage over the Klason method, as it requires a relatively small sample for lignin content estimation.

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls ...