Summary

高分辨率红外热成像(HRIT)的冰核和冰传播的植物研究中的应用

Published: May 08, 2015
doi:

Summary

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

Abstract

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

Introduction

冷冻时植物生长活跃时发生的温度可以是致命的,特别是如果植物具有很少或没有冰冻耐受性。这样的事件霜冻往往会对农业生产造成破坏性影响,也可以在植物自然种群形成群落结构发挥了重要作用,特别是在高山,亚寒带和寒带生态系统1-6。严重的春季霜冻事件在最近几年7-9对水果生产在美国和南美洲的重大影响,并加剧了发病初期温暖的天气其次是较典型的平均气温低。早期的温暖的天气诱使芽突破,激活新枝,叶和花所有这些都很少无霜容忍1,3,10-12增长。这种变化无常的天气模式已经被报道正在发生的气候变化的直接反映,并预计将在FORES一个共同的天气模式eeable未来的13。努力提供经济,有效和环境友好的管理技术或农用化学品,可以提供增加的冰冻耐受性取得了有限的成功为宿主的原因,但是这可以部分地归因于耐冻性和冰冻避免机制在植物的复杂性。 14

霜存活于植物相关联的自适应机制已经传统上被分为两类,耐冻性和冰冻回避。前一类与由一组特定的基因是允许植物容忍与存在和冰在其组织的脱水作用有关的应力的调节生化机制相关联。而在后一类通常是,但不是唯一,与植物中的植物14结构方面确定是否,何时,何地冰形成有关。尽管冻结回避的患病率作为广告aptive机制,一些研究一直致力于在近期了解的底层机制和冻结规避监管。读者可以参考最近15检讨关于这个主题的更详细。

而形成的冰在低温下可能看起来像一个简单的过程中,许多因素有助于确定在哪些冰成核在植物组织和它如何在植物内扩散的温度。如外在和内在冰的存在下成核,异质与均相成核事件,热滞后(防冻剂)的蛋白质,具体糖和其它渗透剂的存在下,和一台主机的植物的结构方面参数都可以发挥显著在植物冷冻过程中的作用。总的来说,这些参数影响在哪个工厂结冰,冰的地方开始以及如何成长的温度。它们也可以影响所得冰晶体的形态。各种方法已被用于研究在植物冷冻过程在实验室条件,包括核磁共振谱(NMR)16,磁共振成像(MRI)17,冷冻显微镜18-19,和低温扫描电子显微镜下(LTSEM )。20冻结在实验室和现场设置整株植物,然而,主要被监测热电偶。使用热电偶来研究冷冻的是基于热解放(熔化焓)当水经历从液体到固体的相变。冷冻,然后被记录作为一个放热事件。21-23即使热电偶是选择在研究中的植物冷冻的典型方法,它们的使用有很大的局限性,限制了冷冻事件期间所获得的信息的量。例如,用热电偶就很难几乎不可能确定在何处开始在植物中的冰,它是如何传播,如果它在传播的速度,甚至,如果某些组织保持无冰。

高分辨率红外热(HRIT)24-27进展,然而,有显著增加,以获取有关整个植物冷冻处理信息的能力,在差分成像模式下使用时尤其如此。28-33在本报告中,我们描述了使用该技术的研究冷冻过程的各个方面,该影响在哪里和以什么样的温度冰在植物中被启动的各种参数。的协议将被呈现,将展示冰核活性(INA)细菌作为一个非本征成核启动在高温,零下温度在一种草本植物冷冻能力, 丁香假单胞菌 (CIT-7)。

高分辨率红外摄像机

该协议和实例本报告中采用红外线高分辨率视频辐射计。所述辐射计( 图1)提供的红外和可见光谱图像和温度数据的组合。照相机的光谱响应是在7.5至13.5微米的范围内,并提供640×480像素的分辨率。所产生的可见光谱图像的内置相机可以与红外图像进行实时,这有利于复杂,热图像的解释是稠合的。透镜用于摄像机A范围可以被用来制造特写和显微镜观察。该相机可以在一个独立的模式下使用,或接口和控制使用的专有软件的笔记本电脑。该软件可以被用来获得各种嵌入在录制的视频热数据。要注意的是各种各样的红外线辐射计是市售的是重要的。因此,至关重要的是,研究者讨论一个知识渊博的产品工程师其预期应用和研究人员测试任何specifi的能力ç辐射计,以提供所需的信息。在所描述的协议中使用的成象辐射计被放置在丙烯酸类盒( 图2)绝缘的聚苯乙烯泡沫塑料I N为了在升温和冷却的协议,以防止暴露于冷凝。无需对所有摄像机或应用这种保护。

Protocol

1.选煤厂材料使用主体植物材料的任叶或整株植物( 玉簪属或菜豆 )。 2.准备水处理解决方案含冰核活性(INA)细菌培养INA的细菌, 丁香假单胞菌 (菌株CIT-7)在培养皿中,在25℃对铜绿琼脂˚F制备按照生产商的方向100%的甘油的10克/升。 后培养已经长大充分,发生在4℃直至需要的,但保持在4℃下进行,以确保冰核活性的高?…

Representative Results

冰+细菌的冰核活动, 丁香假单胞菌 (株CIT-7) 水的10μl降和10微升水含有P.丁香 (CIT-7)被放置于一玉簪叶的背面( 玉簪属)( 图4)。如图所示,对含有冰核细菌水滴冻结第一和负​​责诱导叶冻结而水在叶子表面上的压降保持解冻。 冻结和冰繁殖的木本植物 图5示出冰都开始和冰繁殖…

Discussion

水具有过冷到温度低于0℃,并在该水会冻结可以相当可变温度的能力。36的温度限制为纯水过冷是约-40℃,并且被定义为均相成核点。当水结冰的温度比回暖-40°C它带来的异质存在成核剂,使小冰的胚胎形成,然后作为催化剂,冰的形成和生长。37有众多的自然分子的充当非常有效的冰成核剂,因此大多数的性质的冷冻水发生在温度刚好低于0℃。调节或影响异质成核剂的活性的能…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

该研究是由奥地利科学基金会(FWF):P23681-B16。

Materials

Infrared Camera FLIR SC-660 Many models available depending on application
Infrared Analytical Software FLIR ResearchIR 4.10.2.5 $3,500
Pseudomonas syringae (strain Cit-7) Kindly provided by Dr. Steven Lindow, University of California  Berkeley icelab@berkeley.edu
Pseudomonas Agar F Fisher Scientific DF0448-17-1

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Citazione di questo articolo
Wisniewski, M., Neuner, G., Gusta, L. V. The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants. J. Vis. Exp. (99), e52703, doi:10.3791/52703 (2015).

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