Name: quantification of endogenous auxin and cytokinin during internode culture of ipecac
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Description: Watch this Scientific Journal Video about Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac at JoVE.com

We are grateful to Mr. Akira Murakami of the Department of Applied Biosciences, Toyo University, and Mr. Koudai Taniguchi of the Gunma Agricultural Technology Center for their technical assistance. We are also grateful to Professor Shosaku Kashiwada and Dr. Uma Maheswari Rajagopalan, Toyo University for their suggestions. This study was supported in part by the Research Center for Life and Environmental Sciences, Toyo University.

Note: Ipecac (C. ipecacuanha) was used in this study because it facilitates the analysis of endogenous phytohormones.
1. Growth Conditions to Induce Adventitious Shoots of Ipecac
<ol>
	<li>Prepare phytohormone-free B5 medium adjusted to pH 5.710, and add 0.2% gellan gum. Sterilize by autoclaving.</li>
	<li>Pour 25 mL of the autoclaved medium into a sterile Petri dish (90 mm &#215; 20 mm).</li>
	<li>Cut 8-mm internodal segments of ipecac plantlets using a surgical scalpel with a blade No. 22 on a sterile acrylic plate, and place on the medium (Figure 1).</li>
	<li>Culture on phytohormone-free B5 medium at 24 &#176;C under 15 &#181;mol m&#8722;2 s&#8722;1 of light in a 14-h light/10-h dark cycle for 5 weeks.</li>
	<li>Identify regions I (apical) to IV (basal) in each segment (Figure 1B). Count the number of adventitious shoots &#62; 0.3 mm length in each region under a microscope once a week.</li>
</ol>
2. Extraction and Purification of Phytohormones
<ol>
	<li>Put a 5-mm zirconia bead in each of the four 2-mL sample tubes.</li>
	<li>Cut the segments into regions I to IV (each 2 mm in length) using a surgical scalpel on a sterile acrylic plate.</li>
	<li>Collect eight segments of each region in separate sample tubes (10-30 mg fresh weight).</li>
	<li>Weigh, and then freeze the samples in liquid nitrogen.</li>
	<li>Crush the frozen samples using a bead-based homogenizer.</li>
	<li>Suspend the crushed samples in 1 mL acetonitrile containing 500 pg of each internal standard of auxin and CKs (d5-IAA, d5-tZ, d5-tZR, d6-iP, d6-iPR) using a vortex mixer.</li>
	<li>Hold at 4 &#176;C for 1 h, then centrifuge at 3,500 &#215; g for 5 min at room temperature.</li>
	<li>Wash the pellet in 80% (v/v) acetonitrile containing 1% (v/v) acetic acid. Centrifuge again at 3,500 &#215; g for 5 min at room temperature. Combine the supernatants (from steps 2.7 and 2.8) in a disposable glass tube.</li>
	<li>Add 600 &#181;L water containing 1% (v/v) acetic acid to each combined supernatant, and evaporate the acetonitrile using a vacuum concentrator.</li>
	<li>Equilibrate hydrophilic-lipophilic-balanced (HLB) column cartridges by applying 1 mL each of acetonitrile, methanol, and water containing 1% (v/v) acetic acid.</li>
	<li>Apply one sample solution per equilibrated HLB cartridge.</li>
	<li>Wash the cartridges with 1 mL water containing 1% (v/v) acetic acid.</li>
	<li>Elute all hormones with 2 mL 80% (v/v) acetonitrile containing 1% (v/v) acetic acid in a glass tube.</li>
	<li>Evaporate the acetonitrile in the eluate to obtain extract in water containing 1% (v/v) acetic acid using a vacuum concentrator. 
	NOTE: Do not dry this completely.</li>
	<li>Equilibrate mixed-mode, strong cation-exchange (MCX) column cartridges by applying 1 mL acetonitrile, 1 mL methanol, 0.5 mL 0.1 M HCl, and 1 mL water containing 1% (v/v) acetic acid.</li>
	<li>Apply one sample solution per equilibrated MCX cartridge.</li>
	<li>Wash the cartridges with 1 mL water containing 1% (v/v) acetic acid.</li>
	<li>Elute IAA with 2 mL 30% (v/v) acetonitrile containing 1% (v/v) acetic acid in a glass tube.</li>
	<li>Wash the cartridges with 2 mL 80% (v/v) acetonitrile containing 1% (v/v) acetic acid.</li>
	<li>Wash the cartridges with 2 mL water and 1 mL water containing 5% aqueous ammonia.</li>
	<li>Elute the CKs with 2 mL 60% (v/v) acetonitrile containing 5% aqueous ammonia in a glass tube.</li>
	<li>Evaporate the solvent of each hormone fraction using a vacuum concentrator and store at &#8722;30 &#176;C until the LC-MS/MS analysis.</li>
</ol>
3. LC-MS/MS Analysis of IAA and CKs
<ol>
	<li>Dissolve each hormone extract in a tube with 600 &#181;L of methanol and transfer the solution to a screw neck total recovery vial. Evaporate the solvent using a vacuum concentrator.</li>
	<li>Dissolve the IAA fraction in 20 &#181;L 30% (v/v) acetonitrile, and the CK fractions in 20 &#181;L water containing 1% (v/v) acetic acid in screw neck total recovery vials.</li>
	<li>Analyze the samples in positive ion mode on a triple-quadrupole MS system equipped with an HPLC system. 
	NOTE: We set the HPLC conditions as listed in Table 1.
	<ol>
		<li>For the IAA elution, use a binary gradient of 5%-50% solvent B over 7 min, then increase by 98% solvent B and hold for 1 min, and then equilibrate for 2 min at 5% solvent B by nest injection.</li>
		<li>For the CK elution, use a binary gradient of 2%-40% solvent B over 5 min, 40%-70% solvent B in 7 min, then increase by 95% solvent B and hold for 1 min, and then equilibrate for 2 min at 2% solvent B by nest injection.</li>
		<li>Set the electrospray ionization (ESI)-MS parameters of ion source as listed in Table 2.</li>
	</ol>
	</li>
	<li>Use the multiple reaction monitoring (MRM) transition for quantification of each analyte listed in Table 3.</li>
	<li>Quantify endogenous IAA and CK levels against a standard curve of the ratio of unlabeled to deuterium-labeled standards.</li>
</ol>

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The authors declare that they have no conflicts of interest.

To identify the distribution of phytohormones involved in organogenesis, it is important to use plant materials in which organogenesis can be observed on phytohormone-free medium, because when phytohormones are exogenously applied to explants for inducing shoots or roots, they affect the whole explant, making it difficult to evaluate the inherent plasticity of plants in cell differentiation and organogenesis. Adventitious shoots can be induced on phytohormone-free culture media in other plant species such as Dianthus caryophyllus L.11, Aegle marmelos (L.) Corr&#234;a12, Bacopa monnieri (L.) Pennell13, Celastrus paniculatus Willd.14, and Kalancho&#235; blossfeldiana Poelln.15. It would be possible to apply the protocol in these plant species.
We extracted IAA, tZ, tZR, iP, and iPR in acetonitrile and purified them by solid-phase extraction. The original method uses three types of cartridge columns (HLB, MCX, and weak anion exchange (WAX)) because all phytohormones are purified (including gibberellins, abscisic acid, jasmonic acid, and salicylic acid)16. The HLB column uses a polymeric reverse-phase sorbent, the MCX column uses the same with cation-exchange groups, and the WAX column uses the same with weak anion-exchange groups. The original method elutes CKs (basic) with 60% acetonitrile containing aqueous ammonia on an MCX column in the second step, and then IAA (acidic) with 80% acetonitrile containing 1% acetic acid on a WAX column in the last step. As our focus is auxin and CKs, which interact antagonistically to regulate plant growth17, the simplified protocol uses only the HLB and MCX columns; IAA is eluted with 30% acetonitrile containing 1% acetic acid on the HLB column in the first step. It takes two days from sample preparation to LC-MS/MS analysis.
The acetonitrile solvent should not be allowed to dry out during phytohormone purification in the cartridge columns. If it does, resuspend the sample in acetonitrile to prevent the phytohormones from becoming stuck to the glass tube and lost from the sample. In this protocol, the detection limit with the ion-trap MS system is ~ 10 pg for IAA and CKs from 10-30 mg fresh tissues. To analyze smaller amounts, it would be necessary to collect much more sample or to use MS with higher sensitivity.
Phytohormone analysis is an important technique for the evaluation of plant growth and development. Using this method, we might be able to determine the timing of auxin and CK treatment for adventitious shoot formation in plant species where the optimum culture condition is still unknown. As phytohormone quantification becomes increasingly important, the LC-MS/MS protocol described here will enable the analysis of small samples with high sensitivity and resolution. Our simplification of a previous method will facilitate purification and analysis, and bring high versatility and reproducibility. In the future, this method can be applied to investigate the dynamics of the endogenous auxin and CK during organogenesis in other plant species.

Gottlieb Haberlandt (1854-1945) proposed the concept of &#34;totipotency&#34;, by which plant cells can divide, differentiate, and regenerate whole plants even after their prior differentiation into specific cell types in mature plants1. In tissue culture, whether plant regeneration can be induced or not is determined by the combination and concentration of exogenously applied phytohormones in the growth medium. Skoog and Miller found that adventitious shoots could be induced from tobacco callus on culture medium containing a high ratio of CKs to auxins, whereas adventitious roots could be induced on medium containing a low ratio2. Since that finding, tissue culture has been widely used for the propagation of economically important crops and for the regeneration of transgenic plants3. Adventitious shoots can be induced from tissues other than shoot apical meristem, such as leaves, roots, and internodes. Phytohormone treatment is required for the induction of adventitious shoots in most plant species. However, the optimum concentrations and combinations differ by species and among tissues used as explants. Thus, much effort goes into determining the optimum concentrations and combinations of phytohormones for experiments.
Carapichea ipecacuanha (Brot.) L. Andersson (ipecac) is a medicinal plant that contains alkaloids such as emetine and cephaeline, mainly in the roots4. Root extracts are used as an expectorant, an emetic, and an amoebicide5. Although ipecac grows naturally in the tropical rainforests of Brazil, it is reluctant to set seeds in culture, and the germination rate decreases during seed storage in Japan, with its colder climate6. Instead, it is propagated by tissue culture, in which adventitious shoot formation on internodes is the most efficient method7,8. Interestingly, adventitious shoots can be induced in this species without phytohormone treatment8.
Adventitious shoots are formed on the epidermis in the apical region of internodal segments without callusing, but not in the basal region9. This difference indicates tissue polarity in internodal segments, which is probably under phytohormonal regulation. The ipecac culture system allows a unique opportunity to analyze changes in endogenous phytohormone levels during adventitious shoot formation. Here we introduce our method for the analysis of the endogenous levels of one auxin (indole-3-acetic acid (IAA)) and four CKs (isopentenyl adenine (iP), isopentenyl adenine riboside (iPR), trans-zeatin (tZ), and trans-zeatin riboside (tZR)) in internodal segments through the use of LC-MS/MS.

<table><tbody><tr><td>[2H5]indole-3-acetic acid</td><td>Olchemlm Ltd</td><td>031 1531</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H5]trans-zeatin</td><td>Olchemlm Ltd</td><td>030 0301</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H5]trans-zeatin riboside</td><td>Olchemlm Ltd</td><td>030 0311</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H6]N6-isopentenyl adenine</td><td>Olchemlm Ltd</td><td>030 0161</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H6]N6-isopentenyl adenosine</td><td>Olchemlm Ltd</td><td>030 0171</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>indole-3-acetic acid</td><td>Wako</td><td>098 00181</td><td>standard for LC-MS/MS</td></tr><tr><td>trans-zeatin</td><td>SIGMA-ALDRICH</td><td>Z0876 5MG</td><td>standard for LC-MS/MS</td></tr><tr><td>trans-zeatin riboside</td><td>Wako</td><td>262 01081</td><td>standard for LC-MS/MS</td></tr><tr><td>N6-isopentenyl adenine</td><td>SIGMA-ALDRICH</td><td>D7674 1G</td><td>standard for LC-MS/MS</td></tr><tr><td>N6-isopentenyl adenosine</td><td>ACROS ORGANICS</td><td>22648 1000</td><td>standard for LC-MS/MS</td></tr><tr><td>acetonitrile hypergrade for LC-MS LiChrosolv</td><td>MERCK</td><td>1.00029.1000</td><td>solvent for LC-MS/MS</td></tr><tr><td>Water for chromatography LiChrosolv</td><td>MERCK</td><td>1.15333.1000</td><td>solvent for LC-MS/MS</td></tr><tr><td>HPLC</td><td>SHIMADZU</td><td>Prominence</td><td></td></tr><tr><td>MS</td><td>Sciex</td><td>3200QTRAP</td><td></td></tr><tr><td>Oasis HLB 30 mg/1 cc</td><td>Waters</td><td>WAT094225</td><td>cartridge column</td></tr><tr><td>Oasis MCX 30 mg/1 cc</td><td>Waters</td><td>186000252</td><td>cartridge column</td></tr><tr><td>screw neck total recovery vial</td><td>Waters</td><td>186002805</td><td></td></tr><tr><td>blue, 12 x 32mm screw neck cap and PTFE/silicone septum</td><td>Waters</td><td>186000274</td><td></td></tr><tr><td>Acquity UPLC BEH C18, 2.1x100 mm</td><td>Waters</td><td>186002350</td><td>UPLC column</td></tr><tr><td>Proshell 120 EC-C18, 2.1x50 mm</td><td>Agilent</td><td>699775-902</td><td>UPLC column</td></tr><tr><td>Digital microscope</td><td>Leica</td><td>DHS1000</td><td></td></tr><tr><td>TissueLyser II</td><td>QIAGEN</td><td>85300</td><td></td></tr><tr><td>Surgical blade</td><td>Feather</td><td>No. 22</td><td></td></tr><tr><td>Scalpel handle</td><td>Feather</td><td>No. 4</td><td></td></tr><tr><td>Savant SpeedVac/Refregerated vapor trap</td><td>Thermo Fisher Scientific</td><td>SPD111/RVT4104</td><td>vacuum concentrartor</td></tr><tr><td>Disposable glass tobe (13x100 mm)</td><td>IWAKI</td><td>9832-1310</td><td></td></tr><tr><td>Sterile petri dish</td><td>INA OPTICA</td><td>I-90-20</td><td></td></tr></tbody></table>

quantification of endogenous auxin and cytokinin during internode culture of ipecac

Adventitious shoot formation is an important technique for the propagation of economically important crops and for the regeneration of transgenic plants. Phytohormone treatment is required for the induction of adventitious shoots in most species. Whether adventitious shoots can be induced is determined by the balance between auxin and cytokinin (CK) levels. Much effort goes into determining optimum concentrations and combinations of phytohormones in each tissue used as explants and in each plant species. In ipecac, however, adventitious shoots can be induced on internodal segments in culture medium without phytohormone treatment. This allows the inherent plasticity of ipecac for cell differentiation to be evaluated. To induce adventitious shoots in ipecac, we cultured internodal segments at 24 &#176;C under 15 &#181;mol m&#8722;2 s&#8722;1 of light in a 14-h light/10-h dark cycle on phytohormone-free B5 medium solidified with 0.2% gellan gum for 5 weeks. To investigate phytohormone dynamics during adventitious shoot formation, we measured endogenous indole-3-acetic acid and CKs in the segments by liquid chromatography-tandem mass spectrometry LC-MS/MS. This method allows analysis of endogenous indole-3-acetic acid and CKs levels in a simple manner. It can be applied to investigate the dynamics of endogenous auxin and CK during organogenesis in other plant species.

At the 1st week, no adventitious shoots had formed. At the 2nd week, small shoots appeared. At the 3rd and 4th weeks, the number of shoots increased mostly in the apical regions (I and II) (Figure 2A). At the 5th week, the number of shoots was approximately 7 in region I and 5 in region II (Figure 2B). In contrast, only a few shoots were formed in regions III and IV.
Before culture, the IAA level was slightly higher in region I (4.1 pg/mg, fresh weight (FW)) than in regions II-IV (~ 2.5 pg/mg FW; Figure 3). At the 1st week, the IAA level increased greatly in region IV (11.4 pg/mg FW) and decreased slightly in regions I-III (1.5-2.2 pg/mg FW). At the 2nd week, the IAA level in region IV decreased to ~ 4.4 pg/mg FW, indicating that IAA accumulation in the basal region was transient. By 5 weeks of culture, an IAA concentration gradient emerged, with levels increasing from region I to region IV.
Before culture, there were only trace levels of most CKs (Figure 3). At the 1st week, the tZR level increased to 13.8 pg/mg FW in region II and to 18.1 pg/mg FW in region III. The levels then decreased gradually over 5 weeks of culture. On the other hand, the levels of tZ, iP, and iPR changed only slightly during culture.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56902/56902fig1.jpg" /> 
Figure 1: Preparation of ipecac for adventitious shoot formation. (A) An internodal segment (8 mm long) is cut from regenerated ipecac on a clean bench. (B) The first internode was used for adventitious shoot formation. (C) Segments are cultured on 25 mL phytohormone-free B5 medium in Petri dishes to induce adventitious shoots. Scale bar = 1 cm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56902/56902fig1large.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/56902/56902fig2.jpg" /> 
Figure 2: Distribution of adventitious shoots formed on an internodal segment. (A) Adventitious shoots formed after 0 to 5 weeks of culture. Scale bar = 5 mm. (B) Segments were partitioned into four regions (I-IV), and the number of adventitious shoots in each region was counted at week 5. Data are means &#177; SEM (n = 3). Ten segments were used in each experiment. N.F. = not found. This figure has been modified from Koike et al.9 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56902/56902fig2large.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/56902/56902fig3.jpg" /> 
Figure 3: Time-course analysis of phytohormone levels in internodal segments. Segments were separated into four regions (I-IV). Endogenous IAA and CKs (tZ, tZR, iP, and iPR) in each region were quantified by LC-MS/MS. Because iP and iPR levels were very low, a zoomed graph was inserted inside the same graph. Data are means &#177; SEM (n = 3). Eight segments were used in each experiment. This figure has been modified from Koike et al.9 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56902/56902fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Column</td>
			<td>IAA: ACQUITY BEH C18 &#966;2.1&#215;100 1.7 &#181;m&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td>CKs: Poroshell EC-C18 &#966;2.1&#215;50 2.7 &#181;m</td>
		</tr>
		<tr>
			<td>Temperature&#160;&#160;</td>
			<td>40&#186;C</td>
		</tr>
		<tr>
			<td>Mobile phase</td>
			<td>Solvent A: distilled water + 0.05% acetic acid</td>
		</tr>
		<tr>
			<td></td>
			<td>Solvent B: acetonitrile + 0.05% acetic acid</td>
		</tr>
		<tr>
			<td>Flow rate</td>
			<td>0.35 ml / min</td>
		</tr>
		<tr>
			<td>Injection volume</td>
			<td>18 &#181;l</td>
		</tr>
	</tbody>
</table>
Table 1: HPLC condition in IAA and CK analysis.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Curtain gas (a.u.)*</td>
			<td>10 / 40**</td>
		</tr>
		<tr>
			<td>Collision gas (a.u.)*</td>
			<td>5 / 3**</td>
		</tr>
		<tr>
			<td>Ion spray voltage (V)</td>
			<td>5500</td>
		</tr>
		<tr>
			<td>Temperature (&#186;C)</td>
			<td>600</td>
		</tr>
		<tr>
			<td>Ion source gas 1 (a.u.)*</td>
			<td>30</td>
		</tr>
		<tr>
			<td>Ion source gas 2 (a.u.)*</td>
			<td>40 / 80**</td>
		</tr>
		<tr>
			<td>Resolution</td>
			<td>Unit</td>
		</tr>
	</tbody>
</table>
Table 2: Parameters of ion source. *arbitrary units. **IAA analysis/CK analysis. This table has been modified from Koike et al.9
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>Q1 (m/z)</td>
			<td>Q3 (m/z)</td>
			<td>Declustering potential (V)</td>
			<td>Entrance potential (V)</td>
			<td>Collision energy (V)</td>
		</tr>
		<tr>
			<td>IAA</td>
			<td>176</td>
			<td>130</td>
			<td>26</td>
			<td>6.5</td>
			<td>21</td>
		</tr>
		<tr>
			<td>d5-IAA</td>
			<td>181</td>
			<td>134</td>
			<td>36</td>
			<td>4.0</td>
			<td>23</td>
		</tr>
		<tr>
			<td>tZ</td>
			<td>220</td>
			<td>136</td>
			<td>36</td>
			<td>5.0</td>
			<td>23</td>
		</tr>
		<tr>
			<td>d5-tZ</td>
			<td>225</td>
			<td>137</td>
			<td>31</td>
			<td>5.0</td>
			<td>21</td>
		</tr>
		<tr>
			<td>tZR</td>
			<td>352</td>
			<td>220</td>
			<td>41</td>
			<td>5.5</td>
			<td>23</td>
		</tr>
		<tr>
			<td>d5-tZR</td>
			<td>357</td>
			<td>225</td>
			<td>51</td>
			<td>5.0</td>
			<td>21</td>
		</tr>
		<tr>
			<td>iP</td>
			<td>204</td>
			<td>136</td>
			<td>31</td>
			<td>9.0</td>
			<td>19</td>
		</tr>
		<tr>
			<td>d6-iP</td>
			<td>210</td>
			<td>137</td>
			<td>31</td>
			<td>5.5</td>
			<td>21</td>
		</tr>
		<tr>
			<td>iPR</td>
			<td>336</td>
			<td>204</td>
			<td>31</td>
			<td>5.0</td>
			<td>21</td>
		</tr>
		<tr>
			<td>d6-iPR</td>
			<td>342</td>
			<td>210</td>
			<td>36</td>
			<td>5.5</td>
			<td>21</td>
		</tr>
	</tbody>
</table>
Table 3: MRM transitions of IAA and CKs in LC-MS/MS analysis. This table has been modified from Koike et al.9

Watch this Scientific Journal Video about Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac at JoVE.com

Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

Adventitious shoots can be induced on internodal segments of ipecac without phytohormone treatment. To evaluate phytohormone dynamics during adventitious shoot formation, we measured endogenous auxin and cytokinin in internodal segments by LC-MS/MS.

Adventitious shoot formation is an important technique for the propagation of economically important crops and for the regeneration of transgenic plants. ...

quantification-endogenous-auxin-cytokinin-during-internode-culture

Nanyang Technological University

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JoVE Journal

Environment

9.0K Views.  Toyo University. Onvoorziene scheuten kunnen worden opgewekt op overslagfaciliteiten segmenten van ipecac zonder phytohormone behandeling. Om te beoordelen phytohormone dynamiek tijdens onvoorziene schieten vorming, we gemeten endogene auxine en cytokinine in overslagfaciliteiten segmenten door LC-MS/MS.

Video: Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ripening. Exogenous ethylene applied during early plant development triggers the triple response phenotype; a shorter and thicker hypocotyl with an exaggerated apical hook. Despite the intimate relationship between plants and bacteria, the effect of exogenous ethylene on bacteria has been greatly overlooked. This is partly due to the difficulty of controlling gaseous ethylene within the laboratory without specialized equipment. 2-Chloroethylphosphonic acid (CEPA) is a compound that decomposes into ethylene, chlorine, and phosphate in a 1:1:1:1 molar ratio when dissolved in an aqueous medium of pH 3.5 or greater. Here we describe the use of CEPA to produce in situ ethylene for the investigation of ethylene response in bacteria using the fruit-associated, cellulose-producing bacterium Komagataeibacter xylinus as a model organism. The protocols described herein include both the verification of ethylene production from CEPA via the Arabidopsis thaliana triple response assay and the effects of exogenous ethylene on K. xylinus cellulose production, pellicle properties and colonial morphology. These protocols can be adapted to examine the effect of ethylene on other microbes using appropriate growth media and phenotype analyses. The use of CEPA provides researchers with a simple and efficient alternative to pure ethylene gas for the routine determination of bacterial ethylene response.

The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

Biochemistry

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an Agrobacterium rhizogenes mediated transformation system on pigeon pea. The stem is infected with A. rhizogenes carrying a binary vector, which induced callus after 7 days and adventitious roots 14 days later. The generated transgenic hairy root was identified by morphological analysis and a GFP reporter gene.To further illustrate the application range of this system, CcCIPK14 (Calcineurin B-like protein-interacting protein kinases) was transformed into pigeon pea using this transformation method. The transgenic plants were treated with jasmonic acid (JA) and abscisic acid (ABA), respectively, for the purpose of testing whether CcCIPK14 responds to those hormones. The results demonstrated that (1) exogenous hormones could significantly upregulate the expression levelof CcCIPK14, especially in CcCIPK14 over-expression (OE) plants; (2) the content of Genistein in CcCIPK14-OE lines was significantly higher than the control; (3) the expression level of two downstream key flavonoid synthase genes, CcHIDH1 and CcHIDH2, were up-regulated in the CcCIPK14-OE lines; and (4) the hairy root transgenic system can be used to study metabolically functional genes in non-model plants.

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Here we present an efficient and stable transformation system for the functional analysis of the CcCIPK14 gene as an example, providing a technical basis for studying the metabolism of non-model plants.

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an ...

Genetics

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.

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 ...

Tuning Degradation to Achieve Specific and Efficient Protein Depletion

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting them for degradation. This system is exploited in many non-plant eukaryotes, such that a target protein, tagged with the AID motif, is degraded upon auxin addition. The level of TIR1 expression is critical; excessive expression leads to degradation of the AID-tagged protein even in the absence of auxin, whereas low expression leads to slow depletion. A &#946;-estradiol-inducible AID system was created, with expression of TIR1 under the control of a &#946;-estradiol inducible promoter. The level of TIR1 is tunable by changing the time of incubation with &#946;-estradiol before auxin addition. This protocol describes how to rapidly deplete a target protein using the AID system. The appropriate &#946;-estradiol incubation time depends on the abundance of the target protein. Therefore, efficient depletion depends on optimal timing that also minimizes auxin-independent depletion.

Here, we present a protocol to effectively and specifically deplete a protein of interest in the yeast Saccharomyces cerevisiae using the &#946;-est AID system.

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting ...

Immunology and Infection

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

Biology

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The establishment of an auxin gradient is a key event leading to phototropism and gravitropism. Previously, polar auxin transport (PAT) was shown to establish an auxin gradient in different cellular domains of plants. However, Han et al. recently demonstrated that for proper auxin gradient formation, plasmodesmal callose-mediated symplasmic connectivity between the adjacent cells is also a critical factor. In this manuscript, the strategy to elucidate the role of particular genes, which can affect phototropism and gravitropism by altering the symplasmic connectivity through modulating plasmodesmal callose synthesis, is discussed. The first step is to screen aberrant tropic responses from 3-day-old etiolated seedlings of mutants or over-expression lines of a gene along with the wild type. This preliminary screening can lead to the identification of a range of genes functioning in PAT or controlling symplasmic connectivity. The second screening involves the sorting of candidates that show altered tropic responses by affecting symplasmic connectivity. To address such candidates, the movement of a symplasmic tracer and the deposition of plasmodesmal callose were examined. This strategy would be useful to explore new candidate genes that can regulate symplasmic connectivity directly or indirectly during tropic responses and other developmental processes.

This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The ...

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant productivity and survival. As such, understanding the mechanisms by which stomata develop and pattern has tremendous agronomic value. This paper describes two phenotypic methods using Arabidopsis cotyledons that can be used to characterize the genes controlling stomatal development and patterning. Presented first are procedures for analyzing the stomatal phenotypes using toluidine blue O-stained cotyledons. This method is fast and reliable and does not require the use of epidermal peels, which are widely used for phenotypic analyses but require specialized training. Due to the presence of multiple cysteine residues, the identification and generation of bioactive EPF peptides that have a role in stomatal development have been challenging. Thus, presented second is a procedure used to identify stomatal ligands and monitor their biological activity by bioassays. The main advantage of this method is that it produces reproducible data relatively easily while reducing the amount of peptide solution and the time required to characterize the role of the peptides in controlling stomatal patterning and development. Overall, these well-designed protocols enhance the efficiency of studying the potential stomatal regulators, including cysteine-rich secretory peptides, which require highly complex structures for their activity.

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

This paper describes two phenotyping methods without the use of epidermal peels to characterize the genes controlling stomatal development. The first method demonstrates how to analyze a stomatal phenotype using a toluidine blue O-stained plant epidermis. The second method describes how to identify stomatal ligands and monitor their biological activities.

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant ...

High-Resolution Imaging of Chlorophyll Biosynthesis in Arabidopsis Seedling

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly dynamic process of chlorophyll biosynthesis is triggered during the shift from the dark to the light in flowering plants. At the moment when etiolated seedlings are exposed to the first traces of sunlight, rapid (in order of seconds) conversion of protochlorophyllide into chlorophyllide is mediated by unique light-accepting protein complexes, leading via subsequent metabolic steps to the production of fully functional chlorophyll. Standard techniques for chlorophyll content analysis include pigment extraction from detached plant tissues, which does not apply to studying such fast processes. To investigate chlorophyll kinetics in vivo with high accuracy and spatiotemporal resolution in the first hours after light-induced de-etiolation, an instrument and protocol were developed. Here, we present a detailed procedure designed for statistically robust quantification of chlorophyll in the early stages of Arabidopsis de-etiolation.

Author Spotlight: Non-Invasive High-Resolution Measurement of Chlorophyll Synthesis During De-Etiolation

Here, we describe an advanced tool designed for chlorophyll biosynthesis monitoring during the early stages of Arabidopsis seedling de-etiolation. The novel methodology provides non-invasive real-time chlorophyll fluorescence imaging at high spatial and temporal resolution.

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly ...

Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

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