Name: an induction system for clustered stomata by sugar solution immersion treatment in arabidopsis thaliana seedlings
Uploaded: 2019-02-15T14:30:00
Description: Watch this Scientific Journal Video about An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings at JoVE.com

我们感谢长泽一郎教授对我们工作的善意支持。这项工作得到了日本科学促进会 kakenh给定 17 kh给定和18H05492 的赠款、住友基金会为基础科学研究项目赠款提供的第160.146 笔赠款和佳能基金会向 t. h. h. 提供的赠款。这一实验系统是在 jps kakenh给定号26891006至 k. a. 的财政支持下开发的。我们感谢来自 edanz group (www.edanzediting.com/ac) 的 r比robis, msc 编辑了手稿草稿。

1. 3% 含液半毛拉希盖-skoog 培养基溶液的制备
<ol><li>在烧杯中加入1.1 克的 murashige-skoog 中盐和15克蔗糖。</li>
	<li>加入490毫升蒸馏水, 用搅拌吧搅拌均匀。</li>
	<li>使用 koh 将 ph 值调整到5.8。</li>
	<li>用蒸馏水稀释至500毫升, 并将溶液转移到中等瓶子中。</li>
	<li>通过高压灭菌对溶液进行灭菌 (121°c, 20分钟)。如果不立即使用, 此溶液在灭菌后可保持在4°c。</li>
</ol>2. 含蔗糖介质溶液浸?...

<ol>
	<li>Raschke, K., Fellows, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stomatal+movement+in+Zea+mays:+shuttle+of+potassium+and+chloride+between+guard+cells+and+subsidiary+cells.">Stomatal movement in Zea mays: shuttle of potassium and chloride between guard cells and subsidiary cells.</a> Planta. 101 (4), 296-316 (1971).</li><li>Higaki, T., Hashimoto-Sugimoto, M., Akita, K., Iba, K., Hasezawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+and+environmental+responses+of+PATROL1+in+Arabidopsis+subsidiary+cells.">Dynamics and environmental responses of PATROL1 in Arabidopsis subsidiary cells.</a> Plant and Cell Physiology. 55 (4), 773-780 (2013).</li><li>Bergmann, D. C., Sack, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stomatal+development.">Stomatal development.</a> Annual Review of Plant Biology. 58, 163-181 (2007).</li><li>Pillitteri, L. J., Torii, K. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+stomatal+development.">Mechanisms of stomatal development.</a> Annual Review of Plant Biology. 63, 591-614 (2012).</li><li>Sakai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemical+compound+bubblin+induces+stomatal+mispatterning+in+Arabidopsis+by+disrupting+the+intrinsic+polarity+of+stomatal+lineage+cells.">The chemical compound bubblin induces stomatal mispatterning in Arabidopsis by disrupting the intrinsic polarity of stomatal lineage cells.</a> Development. 144 (3), 499-506 (2017).</li><li>Akita, K., Hasezawa, S., Higaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+of+plant+stomatal+one-cell-spacing+rule+by+sugar+solution+immersion.">Breaking of plant stomatal one-cell-spacing rule by sugar solution immersion.</a> PLOS One. 8 (9), 72456 (2013).</li><li>Chen, X. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+callose+synthase+gene+GSL8+is+required+for+cytokinesis+and+cell+patterning.">The Arabidopsis callose synthase gene GSL8 is required for cytokinesis and cell patterning.</a> Plant Physiology. 150 (1), 105-113 (2009).</li><li>Guseman, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+cell-to-cell+connectivity+and+stomatal+patterning+by+loss-of-function+mutation+in+Arabidopsis+chorus+(glucan+synthase-like+8).">Dysregulation of cell-to-cell connectivity and stomatal patterning by loss-of-function mutation in Arabidopsis chorus (glucan synthase-like 8).</a> Development. 137 (10), 1731-1741 (2010).</li><li>Akita, K., Hasezawa, S., Higaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+microtubules+and+fusicoccin+response+in+clustered+stomatal+guard+cells+induced+by+sucrose+solution+immersion.">Cortical microtubules and fusicoccin response in clustered stomatal guard cells induced by sucrose solution immersion.</a> Plant Signaling and Behavior. 13 (4), 1454815 (2018).</li><li>Akita, K., Hasezawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sugar+solution+induces+clustered+lips.">Sugar solution induces clustered lips.</a> Cytologia. 79 (2), 125-126 (2014).</li><li>Holzinger, A., Buchner, O., L&#252;tz, C., Hanson, M. 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(106), 53437 (2015).</li></ol>

我们提出了用含蔗糖介质溶液浸没处理诱导刺五幼苗聚集气孔的方案。如图所示, 这种方法是非常简单的, 不需要专门的技能, 但可以有效地诱导聚集气孔。超过45% 的保护细胞聚集在3% 含蔗糖的介质溶液中 (平均值超过20个独立观察)6。此外, 该实验系统可直接应用于转基因或突变线, 如表达 ct-gfp (图 1) 或 gfp-tub6 (图 2) 所示。虽然?...

植物气孔是光合作用和蒸腾作用气体交换的重要器官, 气孔运动是通过离子驱动的吸水和释放来实现的。在显微镜下, 我们可以观察到在叶片和茎的表面上气孔的间隔分布模式。气孔的这种间隔分布被认为有助于气孔运动, 这是由离子和水交换之间的保护细胞和邻近的表皮细胞1,2。聚集气孔的实验诱导系统可用于研究气孔间隔分布的重要性。
据报道, 保护细胞分化 3、4或使用化合物5处理的关键基因的基因修饰可以诱导气孔的空间聚集.我们还报告说, 浸泡治疗与中等溶液补充糖, 包括蔗糖, 葡萄糖, 果糖引起气孔聚集在拟南芥幼苗的子叶 6.在蔗糖处理的子叶表皮中观察到分离子列表和表皮细胞的新细胞壁中的钙质减少, 这表明蔗糖溶液浸泡治疗对细胞壁有负面影响, 从而防止了细胞壁的泄漏, 并防止了细胞壁的渗漏。保护细胞分化 (如转录因子) 对邻近表皮细胞的关键基因产物的异位作用6。通过对gsl8/突变体7、8的研究, 提出了类似的机制。我们的利用含蔗糖介质溶液对聚集气孔进行重复诱导的实验系统是一种相当简单、廉价的实验系统。它也可以用来研究细胞内的结构, 如细胞器和细胞骨架聚集保护细胞时, 应用于转基因线表达荧光标记, 标签细胞内结构9,10个。

<table border="0" cellpadding="0" cellspacing="0" style="width:685px;" width="686">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">24-well plate</td>
			<td style="width:135px;">Sumitomo Bakelite</td>
			<td style="width:168px;">MS-0824R</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">488 nm laser</td>
			<td style="width:135px;">Furukawa Denko</td>
			<td style="width:168px;">HPU-50101-PFS2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">488 nm laser</td>
			<td style="width:135px;">Olympus</td>
			<td>Sapphire488-20/O</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">510 nm long-pass filter</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">BA510IF</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">524 - 546 nm band-pass filter</td>
			<td style="width:135px;">Semrock</td>
			<td style="width:168px;">FF01-535/22-25</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">530 nm short-pass filter</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">BA530RIF</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">561 nm laser</td>
			<td style="width:135px;">CVI Melles Griot</td>
			<td style="width:168px;">85-YCA-025-040</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">604 - 644 nm band-pass filter</td>
			<td style="width:135px;">Semrock</td>
			<td style="width:168px;">FF01-624/40-25</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Confocal laser scanning head</td>
			<td style="width:135px;">Yokogawa</td>
			<td style="width:168px;">CSU10</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Confocal laser scanning head</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">FV300</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Cooled CCD camera</td>
			<td style="width:135px;">Photometrics</td>
			<td style="width:168px;">CoolSNAP HQ2</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Image acquisition software</td>
			<td style="width:135px;">Molecular Devices</td>
			<td style="width:168px;">MetaMorph version 7.8.2.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Image acquisition software</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">FLUOVIEW v5.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Immersion oil</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">Immersion Oil Type-F</td>
			<td>ne = 1.518 (23 degrees)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Inverted microscope</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">IX-70</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Inverted microscope</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">IX-71</td>
			<td></td>
		</tr>
		<tr height="180">
			<td height="180" style="height:180px;width:216px;">Murashige and Skoog Plant Salt Mixture</td>
			<td style="width:135px;">FUJIFILM Wako Pure Chemical Corporation</td>
			<td style="width:168px;">392-00591</td>
			<td style="width:167px;">Murashige T and Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15(3), 473-497.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Objective lens&#160;</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">UPlanApo 100x / 1.35 NA Oil Iris 1.35</td>
			<td>NA = 1.35</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Objective lens&#160;</td>
			<td style="width:135px;">Olympus</td>
			<td style="width:168px;">UPlanAPO 40x / 0.85 NA</td>
			<td>NA = 0.85</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Sucrose</td>
			<td style="width:135px;">FUJIFILM Wako Pure Chemical Corporation</td>
			<td style="width:168px;">196-00015</td>
			<td></td>
		</tr>
	</tbody>
</table>

an induction system for clustered stomata by sugar solution immersion treatment in arabidopsis thaliana seedlings

气孔运动介导植物气体交换, 这是光合作用和蒸腾作用所必需的。气孔开口和关闭分别是通过保护细胞体积的显著增加和减少来完成的。由于在气孔运动过程中, 离子和水的穿梭迁移发生在保护细胞和较大的相邻表皮细胞之间, 因此植物气孔的间隔分布被认为是气孔运动的最佳分布。改变气孔间距图案的实验系统有助于检验气孔间距图案的意义。几个关键基因与间隔气孔分布已被确定, 聚集气孔可以通过改变这些基因实验诱导。另外, 外源性处理也可以诱导聚集气孔, 而不进行基因改造。本文介绍了一种简单的拟南芥幼苗聚集气孔的诱导系统, 该系统采用含蔗糖介质溶液浸没处理。我们的方法简单, 直接适用于转基因或突变线。较大的叶绿体被认为是蔗糖诱导的聚集保护细胞的细胞生物学特征。此外, 皮质微管的一个具有代表性的共聚焦显微图像显示为聚集保护细胞的细胞内观察的一个例子。在控制条件下, 在聚集保护细胞中保持皮质微管的径向方向, 就像在间隔的保护细胞中一样。

本文提出了一种简单的方法, 在a. thaliana幼苗中, 用含蔗糖的介质溶液诱导气孔聚集的方法。在含蔗糖的介质溶液中生长的聚集保护细胞 (图 1b) 的叶绿体大于在无蔗糖控制条件下生长的保护细胞 (图 1a)。叶绿体的扩大得到了叶绿体标记 ct-gfp11和叶绿素自体荧光 (图...

Watch this Scientific Journal Video about An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings at JoVE.com

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

本方案的目的是论证如何通过含糖培养基溶液浸泡处理诱导拟南芥幼苗子叶聚集气孔, 以及如何观察叶绿体等细胞内结构和微管在聚集保护细胞使用共聚焦激光显微镜。

拟南芥幼苗糖溶液浸入治疗的聚集性植物诱导系统

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a ...

该方法回答了植物发育生物学的一个关键问题。也就是说，斯托马塔的空间分布的重要性是什么？我们的诱导系统，用于聚类的波托塔使用蔗糖溶液是相当容易和直接适用于转基因或突变线的阿拉伯多普西沙利纳。首先，在烧杯中加入1.1克Murashige-Skoog中盐和15克蔗糖。在烧杯中加入490毫升蒸馏水，与搅拌棒混合。使用氢氧化钾将pH调至5.8。然后用500毫升蒸馏水稀释溶液，将稀释溶液转移到中瓶中。在此之后，自动解保存解决方案。根据文本协议准备灭菌溶液。然后，在无菌条件下工作，将携带荧光标记的大约50个转基因 A.thaliana 种子放入1.5毫升管中。接下来，在管子中加入一毫升70%乙醇，通过倒置管子五次混合。让种子沉入管底后，用微管轻轻取出乙醇。在种子中加入一毫升灭菌溶液，将管子倒置五次混合。用微管轻轻从种子中去除灭菌溶液。然后加入一毫升的无菌水。在24孔板的每个孔中加入1.5毫升的Murashige-Skoog溶液。然后在每个井中加入两个灭菌种子。使用两层副膜，将盖子胶带固定到盘子上。接下来，将板转移到生长室孵育14天。首先，将 30 微升 Murashige-Skoog 溶液从 24 孔板转移到玻璃滑梯的中心。使用解剖剪刀，从14天大的幼苗中取出科蒂冬。漂浮在科蒂莱顿与观察侧面对穆拉希格 - 斯科格解决方案的下降。根据《可视化实验杂志》前面描述的方法，准备科蒂顿。将试样放在共合激光显微镜的舞台上。最后，使用亮场照明选择聚类防护单元进行观察。在此协议中，在阿.塔利亚纳幼苗中诱导了口腔聚类。在含有介质的蔗糖中生长的聚类保护细胞比在无蔗糖条件下生长的防护细胞具有更大的叶绿体。叶绿体肿的扩大用叶绿体频闪标记物和叶绿素自荧光素得到确认，这表明蔗糖处理导致叶绿体中淀粉颗粒积累。此外，GFP浴缸-6的共生观察显示，皮质微管在蔗糖处理的细胞中是径向的，就像在无蔗糖保护细胞中一样。实际上，我们偶然发现了这种方法。发现后，我们研究了聚类斯托马塔的环境反应。我们相信我们的系统可以帮助研究原子体分布的重要性。

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An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

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Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

农业经营条件下土壤侵蚀和沉积与土壤碳酸盐浓度关系的测量与制图

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

A Novel Method for the Pentosan Analysis Present in Jute Biomass and Its Conversion into Sugar Monomers Using Acidic Ionic Liquid

利用酸性离子液体戊聚糖分析黄麻生物量及其转化为糖单体的新方法

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

一种灵活的低成本水培系统在无菌条件下评价植物对小分子的响应

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

美国食品药品监督管理局追踪者在拟南芥底部水槽组织中的射击运动

The symplastic tracer 5(6)-carboxyfluorescein diacetate (CFDA) has been widely applied in living plants to demonstrate the intercellular connection, ...

Cortisol Extraction from Sturgeon Fin and Jawbone Matrices

皮质醇从鱼翅和爪骨基质提取

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) ...

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

卡诺哈布迪蒂斯精英热休克反应的测量诱导标准化方法

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, ...

Using Changes in Leaf Transmission to Investigate Chloroplast Movement in Arabidopsis thaliana

Using Changes in Leaf Transmission to Investigate Chloroplast Movement in Arabidopsis thaliana

利用叶片传播的变化来研究拟南芥的叶绿体运动

Chloroplast movement in leaves has been shown to help minimize photoinhibition and increase growth under certain conditions. Much can be learned about ...