我们感谢东洋大学应用生物科学系的 Koudai 先生和群马县农业技术中心谷口先生的技术援助。我们还感谢 Shosaku Kashiwada 教授和东洋大学 Maheswari Rajagopalan 博士的建议。这项研究由东洋大学生命与环境科学研究中心部分支持。

注: 本研究使用催吐剂 (ipecacuanha), 因为它有助于分析内源性激素。
1. 诱导催吐剂不定芽的生长条件
<ol><li>制备无激素的 B5 培养基, 调整为 pH 值为 5.710, 并添加0.2% 结冷胶胶。热处理消毒。</li>
	<li>将25毫升的蒸压介质倒入无菌培养皿 (90 毫米 x 20 毫米)。</li>
	<li>在不育的丙烯酸板上, 用一把22刀片的外科手术刀切开8毫米茎的催吐剂苗, 并放置在培养基上 (图 1)。</li>
	<li>无激素 B5 培养基的培养在24摄氏度以下15µmol m−2 s−1的光在14小时 light/10-h 黑暗周期5周。</li>
	<li>在每个段中标识区域 I (顶端) 到 IV (基底) (图 1B)。每周一次显微镜下计算每个区域中不定芽的数量 > 0.3 毫米长度。</li>
</ol>2. 激素的提取和纯化
<ol><li>在四2毫升的样品管中放入5毫米氧化锆珠。</li>
	<li>在不育的丙烯酸板上使用外科手术刀将片段切成区域 I 至 IV (每长度2毫米)。</li>
	<li>收集八段的每个区域在不同的取样管 (10-30 毫克新鲜重量)。</li>
	<li>称重, 然后冻结液氮中的样品。</li>
	<li>用珠基均质机粉碎冷冻样品。</li>
	<li>在1毫升乙腈中悬浮被粉碎的样品, 其中含有生长素和中正的每个内部标准的 500 pg (d5-IAA, d5-, d5-tZR, d6-iP, d6-知识产权) 使用涡旋混合器。</li>
	<li>保持在4°c 为1小时, 然后离心机在 3500 x g在室温下5分钟。</li>
	<li>在含有 1% (v/v) 乙酸的 80% (v/v) 乙腈中清洗颗粒。离心机再次在 3500 x g室温下5分钟。将上清液 (从步骤2.7 和 2.8) 组合在一次性玻璃管中。</li>
	<li>添加600µL 含 1% (v/v) 乙酸的水, 每联合上清, 并蒸发的乙腈使用真空选矿厂。</li>
	<li>平衡亲水性-亲脂平衡 (HLB) 柱筒, 适用于每乙腈, 甲醇和含 1% (钒/钒) 乙酸的水的1毫升。</li>
	<li>每平衡 HLB 墨盒应用一个样本解决方案。</li>
	<li>用含有 1% (v/v) 醋酸的1毫升水清洗墨盒。</li>
	<li>洗脱所有激素与2毫升 80% (v/v) 乙腈含有 1% (v/v) 乙酸在玻璃管。</li>
	<li>将洗脱液中的乙腈蒸发, 用真空浓缩器在含有 1% (v/v) 乙酸的水中获得萃取物。 注: 不要完全干燥。</li>
	<li>平衡混合模式, 强阳离子交换 (MCX) 柱盒通过应用1毫升乙腈, 1 毫升甲醇, 0.5 毫升0.1 米 HCl, 和1毫升水含 1% (v/v) 乙酸。</li>
	<li>每个平衡 MCX 墨盒应用一个示例解决方案。</li>
	<li>用含有 1% (v/v) 醋酸的1毫升水清洗墨盒。</li>
	<li>洗脱 IAA 与2毫升 30% (v/v) 乙腈含有 1% (v/v) 乙酸在玻璃管。</li>
	<li>用2毫升 80% (v/v) 乙腈清洗墨盒, 含 1% (v/v) 乙酸。</li>
	<li>用2毫升水和1毫升水清洗墨盒, 含5% 水氨。</li>
	<li>洗脱中正与2毫升 60% (v/v) 乙腈含有5% 水氨的玻璃管。</li>
	<li>使用真空浓缩器和存储在−30°c, 直到 LC-ms/毫秒分析, 蒸发每个荷尔蒙分数的溶剂。</li>
</ol>3. IAA 和中正的 LC-ms/毫秒分析
<ol><li>将每个激素提取物溶解在600µL 甲醇的管中, 将溶液转移到螺钉颈部总回收瓶中。使用真空浓缩器蒸发溶剂。</li>
	<li>将20µL 30% (v/v) 乙腈中的 IAA 分数和20µL 含 1% (v/v) 乙酸的 CK 分数溶解在螺钉颈总回收瓶中。</li>
	<li>以高效液相色谱系统为例, 分析了三元四极 MS 系统正离子模式下的样品。 注意: 我们设置了表 1中列出的高效液相色谱条件。<ol>
<li>对于 IAA 洗脱, 使用 5%-50% 溶剂 b 的二进制梯度超过7分钟, 然后增加98% 溶剂 b 和保持1分钟, 然后平衡2分钟在5% 溶剂 b 通过巢注射。</li>
		<li>对于 CK 洗脱, 使用 2%-40% 溶剂 b 的二进制梯度超过5分钟, 40%-70% 溶剂 b 在7分钟, 然后增加95% 溶剂 b 和举行1分钟, 然后平衡2分钟在2% 溶剂 b 通过巢注射。</li>
		<li>设置离子源的电喷雾电离 (ESI)-MS 参数, 如表 2所示。</li>
	</ol>
</li>
	<li>使用多重反应监测 (MRM) 转换来量化表 3中列出的每个分析物。</li>
	<li>将内源 IAA 和 CK 水平与未标记比率与氘标记标准的标准曲线进行量化。</li>
</ol>

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作者声明他们没有利益冲突。

为了确定参与器官激素的分布, 重要的是使用植物材料在无激素培养基上观察器官, 因为当激素被外部应用于外植体诱导芽或根, 它们影响整个外植体, 使得很难评价植物的内在可塑性的细胞分化和器官。不定芽可诱导其他植物的无激素培养基 (如石竹石竹L.11、 Aegle marmelos (l.) Corrêa12、 Bacopa 床子(l.) Pennell13, 藤苋菜。<s...

Haberlandt (1854-1945) 提出了 "全能" 的概念, 在成熟植物中, 植物细胞可以分化、分化和再生整个植物, 甚至在它们的前向特定细胞类型的差异1。在组织培养中, 能否诱导植物再生是由外部在生长培养基中应用激素的组合和浓度决定的。Skoog 和米勒发现, 在含有高中正比植物生长素的培养基上, 烟草愈伤组织可诱导不定芽, 而不定根可在含有低比值2 的培养基上诱导。自此发现以来, 组织培养已广泛用于经济重要作物的繁殖和转基因植物的再生3。不定芽可诱导从其他组织以外的茎尖分生, 如叶, 根, 和节间。在大多数植物中诱导不定芽需要激素治疗。然而, 最佳的浓度和组合不同的物种和组织之间作为外植体。因此, 大量的努力去确定激素的最佳浓度和组合的实验。
Carapichea ipecacuanha(Brot)l. 安德森 (催吐剂) 是一种药用植物, 含有吐根碱和 cephaeline 等生物碱, 主要在根4 中。根提取物用作祛痰、催吐剂和 amoebicide5。虽然催吐剂自然生长在巴西的热带雨林中, 但它不愿意在文化中设置种子, 在日本的种子储藏过程中, 发芽率下降, 气候较冷的6。相反, 它传播的组织培养, 在节间不定的拍摄形成是最有效的方法7,8。有趣的是, 不定芽可以诱导在这个物种没有激素治疗8。
不定芽形成于茎节顶端区域的表皮上, 不愈伤组织, 但不在基底区域9。这种差异表明茎段的组织极性, 这可能是在 phytohormonal 调节。催吐剂培养系统允许一个独特的机会来分析在不定芽形成期间内源激素水平的变化。在这里, 我们介绍了分析一个生长素 (indole-3-acetic 酸 (IAA)) 和四中正 (isopentenyl 腺嘌呤 (iP), isopentenyl 腺嘌呤核苷 (iPR),反式-玉米 () 和反式-玉米核苷 (tZR) 的内生水平的方法。在茎段中通过使用 LC/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

不定芽形成是重要的技术, 对重要作物的繁殖和转基因植物的再生。在大多数物种中诱导不定芽需要激素治疗。不定芽能否诱发是由生长素与细胞分裂素 (CK) 水平的平衡决定的。大量的努力, 以确定最佳的浓度和组合激素在每个组织作为外植体和在每个植物物种。在催吐剂中, 无激素处理可诱导培养基中茎段的不定芽。这使得催吐剂的内在可塑性可以被评估。为了诱导催吐剂中的不定芽, 我们在15µmol m−2 s−1的茎段中培养了24摄氏度, 在14小时 light/10-h 黑循环中, 在无激素 B5 培养基上固化0.2% 结冷胶胶5周。通过液相色谱-串联质谱 (LC ms) 测定了不定芽形成过程中的激素动态, 并对 indole-3-acetic 中的内源性酸和中正进行了检测。这种方法可以简单地分析内源性 indole-3-acetic 酸和中正水平。可用于研究其他植物器官中内源生长素和 CK 的动态变化。

在 1st周, 没有形成不定芽。在 2nd周, 出现了小芽。在 3rd和 4th周, 芽数增加主要在顶端区域 (I 和 II) (图 2A)。在 5th周, 芽的数量在区域 i 和 5 (图 2B) 中大约为7。相比之下, 第三和 iv. 区只形成了几枝芽。
在培养之前, IA...

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Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

催吐剂节间培养中内源生长素和细胞分裂细胞的量化

无激素治疗的催吐剂茎段可诱发不定芽。为了评价不定芽形成过程中的激素动态, 我们用 LC-ms/ms 测定了茎段内源性生长素和细胞分裂素。

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

9.0K 次观看.  Toyo University. 无激素治疗的催吐剂茎段可诱发不定芽。为了评价不定芽形成过程中的激素动态, 我们用 LC-ms/ms 测定了茎段内源性生长素和细胞分裂素。

视频: 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.

利用乙烯释放化合物，2-氯乙基膦酸，作为一种工具来研究细菌乙烯反应

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

科研

生物化学

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 ...

遗传学

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.

使用SAX-HPLC从不同植物物种中提取和定量可溶性、放射性标记肌醇多磷酸盐

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.

调整降解以实现特定和高效的蛋白质消耗

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

免疫与感染

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.

侧根诱导系统<em&gt;拟南芥</em&gt;和玉米

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

生物学

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.

的策略，以验证胼胝质介导的Plasmodesmal浇注在热带反应中的作用

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.

用于植物/微生物分子相互作用和信号的同时系统分析的水培共培养系统

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.

分离块茎原生质体流式细胞仪测量 Endoreduplication 的研究

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.

通过表皮表型评分 鉴定 参与气孔发育的基因

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 ...

拟南芥幼苗中叶绿素生物合成的高分辨率成像

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.

作者聚焦:去硫醇化过程中叶绿素合成的无创高分辨率测量

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.

拟南芥脱硫早期叶绿素生物合成动力学测定的非侵入性测定

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

催吐剂节间培养中内源生长素和细胞分裂细胞的量化

摘要

探索更多视频

此视频中的章节

利用乙烯释放化合物，2-氯乙基膦酸，作为一种工具来研究细菌乙烯反应

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

使用SAX-HPLC从不同植物物种中提取和定量可溶性、放射性标记肌醇多磷酸盐

调整降解以实现特定和高效的蛋白质消耗

侧根诱导系统

的策略，以验证胼胝质介导的Plasmodesmal浇注在热带反应中的作用

用于植物/微生物分子相互作用和信号的同时系统分析的水培共培养系统

分离块茎原生质体流式细胞仪测量 Endoreduplication 的研究

通过表皮表型评分鉴定参与气孔发育的基因

拟南芥脱硫早期叶绿素生物合成动力学测定的非侵入性测定

催吐剂节间培养中内源生长素和细胞分裂细胞的量化

摘要

探索更多视频

此视频中的章节

利用乙烯释放化合物，2-氯乙基膦酸，作为一种工具来研究细菌乙烯反应

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

使用SAX-HPLC从不同植物物种中提取和定量可溶性、放射性标记肌醇多磷酸盐

调整降解以实现特定和高效的蛋白质消耗

侧根诱导系统

的策略，以验证胼胝质介导的Plasmodesmal浇注在热带反应中的作用

用于植物/微生物分子相互作用和信号的同时系统分析的水培共培养系统

分离块茎原生质体流式细胞仪测量 Endoreduplication 的研究

通过表皮表型评分 鉴定 参与气孔发育的基因

拟南芥脱硫早期叶绿素生物合成动力学测定的非侵入性测定

通过表皮表型评分鉴定参与气孔发育的基因