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Nov 2020

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Dark Respiration Measurement from Arabidopsis Shoots
拟南芥芽的暗呼吸测量   

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Abstract

Dark respiration refers to experimental measures of leaf respiration in the absence of light, done to distinguish it from the photorespiration that occurs during photosynthesis. Dark aerobic respiration reactions occur solely in the mitochondria and convert glucose molecules from cytoplasmatic glycolysis and oxygen into carbon dioxide and water, with the generation of ATP molecules. Previous methods typically use oxygen sensors to measure oxygen depletion or complicated and expensive photosynthesis instruments to measure CO2 accumulation. Here, we provide a detailed, step-by-step approach to measure dark respiration in plants by recording CO2 fluxes of Arabidopsis shoot and root tissues. Briefly, plants are dark acclimated for 1 hour, leaves and roots are excised and placed separately in airtight chambers, and CO2 accumulation is measured over time with standard infrared gas analyzers. The time-series data is processed with R scripts to produce dark respiration rates, which can be standardized by fresh or dry tissue mass. The current method requires inexpensive infrared gas analyzers, off-the-shelf parts for chambers, and publicly available data analysis scripts.

Keywords: NFS1 (NFS1), Dark respiration (暗呼吸), CO2 (二氧化碳), Mitochondria (线粒体), Flux (通量)

Background

Plants produce energy in the form of ATP from glucose oxidation through mitochondrial respiration (Glucose + oxygen = carbon dioxide + water). In order to differentiate from photorespiration inherent to photosynthesis, dark respiration measurements are made in the absence of light (Graham, 1980). Mitochondria are essential organelles involved in energy production, amino acid biosynthesis, iron-sulfur (Fe-S) cluster cofactor biosynthesis, and other metabolic pathways that affect growth and stress responses (Lill, 2009). We identified two mitochondria-localized Fe-S cluster proteins, NITROGEN FIXATION S-LIKE1 (NFS1) and its interactor FRATAXIN (FH), as novel players in plant defense responses (Fonseca et al., 2020). In the current manuscript, we describe the methods used in this previous research to test a possible effect of the nfs1 and fh mutants, and overexpression lines, on mitochondrial respiration using measurements of dark respiration of the entire shoot and root. This simple protocol can be performed using plants grown entirely in controlled conditions in growth chambers. It involves the flux measurement of the dark respiration by-product CO2 from tissues in small airtight chambers, using an LI-850 CO2/H2O gas analyzer (LICOR) in a dark room with a supplemental photosynthetically inactive yellow LED light. The CO2 flux for each plant is measured during a 2 min 30 s interval using the software LI-8x0 v1.02 (LICOR). Specific shoot and root respiration rates (nmol CO2 gfw-1 s-1) are calculated from the raw CO2 flux data using R (v.3.6.0; https://www.r-project.org/), where linear regression is used to calculate the CO2 flux rate with the dead band set at 60 s, and then divided by total shoot fresh weight.


Prior descriptions of plant respiration measurements often included complex apparatus consisting of independent equipment to move air and measure CO2 with an infrared gas analyzer, and using a mass flow controller (Martin et al., 1981; Condori-Apfata et al., 2019). More recently, photosynthesis instruments that are an order of magnitude more expensive than the gas analyzer used here have been used with custom modifications (Tomaz et al., 2010). As an alternative to measuring the production of CO2, other protocols make use of oxygen sensors to instead measure O2 depletion (Li et al., 2013). In comparison, we have accomplished the same goals using less expensive and relatively less complex off-the-shelf parts that can be assembled and used by any plant laboratory. In addition, we have made the R scripts for data analysis publicly available to ensure the reproducibility of the method.


This protocol will be useful for researchers who need to measure dark respiration as a proxy for changes in mitochondrial abundance and/or activity, as we have done inFonseca et al. (2020). However, respiration has also been hypothesized to be a potential target for increasing metabolic efficiency, as has been done for photosynthesis (Amthor et al., 2019). Simulation results highlight the opportunity to target respiration as an unappreciated avenue for crop improvement, where both leaf and root respiration need to be considered for the whole-plant carbon allocation analysis (Holland et al., 2020). Indeed, a similar protocol as outlined here was used to measure in-light root respiration of 1,104 wheat seedlings to conduct a genome wide association study (Guo et al., 2021). This work found substantial heritable variation for specific root respiration as well as candidate genes responsible for that variation. Bunce (2021) showed that CO2 depletion methods for respiration closely match the total mass reduction estimates of respiration, validating the approach. Therefore, this reproducible protocol can have an impact on multiple fields of plant biology, ranging from understanding the molecular basis of respiration to study respiration as a breeding target.

Materials and Reagents

  1. Falcon 6-well clear flat bottom plate (Corning, catalog number: 353224)

  2. Petri plates (15 × 100 mm) (Carolina, catalog number: 741251)

  3. 7.5 cm3 internal volume opaque PVC pipe with threaded ends (United States Plastic Corp, 1/4" × 6" Schedule 80 CPVC Nipple, catalog number: 30207)

  4. Threaded cap with 1/8” hole drilled in the center (United States Plastic Corp, 1/4" FNPT Nylon Threaded Cap, catalog number: 62164)

  5. Quick-connect male and female fittings (LI-COR, Inc., catalog numbers: 300-07124 and 300-07125)

  6. Balston filter (LI-COR, Inc., catalog number: 300-01961)

  7. Rubber seals (LI-COR, Inc., catalog number: 167-07256)

  8. Parafilm M laboratory film (Amazon, catalog number: PM999)

  9. Bev-A-Line 1/4'' OD 15 m tubing (LI-COR, Inc., catalog number: 8150-250)

  10. Quick connect, female bulkhead fitting (LI-COR, Inc., catalog number: 300-07126)

  11. Quick connect, plug bulkhead (LI-COR, Inc., catalog number: 300-07127)

  12. Quick connect straight union (LI-COR, Inc., catalog number: 300-03123)

  13. Aluminum foil (Amazon, any brand)

  14. Drierite (LI-COR, Inc., catalog number: 622-04299)

  15. Arabidopsis seeds (ABRC., https://abrc.osu.edu)

  16. Murashige & Skoog (MS) media with vitamins (Grainger, catalog number: M70800-50.0)

  17. Sucrose (VWR, catalog number: BDH9308-500G)

  18. Potassium hydroxide (KOH) (VWR, catalog number: BDH9262-500G)

  19. Phytagel (Sigma-Aldricht, catalog number: 71010521)

  20. Ethanol absolute 100% (VWR, catalog number: 71006012)

  21. Triton X-100 (Fischer scientific, catalog number: AC327372500)

  22. Sodium Hypochlorite 8% (Clorox Disinfecting Bleach, catalog number: CLO32260)

  23. 1/2 MS liquid media pH 5.7 (see Recipes)

  24. Bleach solution (see Recipes)

Equipment

  1. Orbital Shaker (Thermolyne Bigger Bill, catalog number: M49235)

  2. LI-850 CO2/H2O Analyzer (LI-COR, Inc. catalog number:)

  3. Computer with Windows 10 and USB ports to run LI-COR software

  4. Bead bath (Lab Armor, catalog number: 74300-714)

  5. Sterile cabinet (Thermo Scientific, catalog number: 13-261-221)

  6. Autoclave (Steris Amsco Century, catalog number: SG-120)

  7. Metallic beads (Lab Armor, catalog number: 42370-004)

  8. Timer/stopwatch (Big Digit Timer, catalog number: ML5004)

  9. Compressed air duster (Blow Off Duster, catalog number: 152-112-226)

  10. Laboratory balance scale (Mettler Toledo, catalog number: ME103TE)

  11. Curved tip forceps for plant handling (Epredia, catalog number: 1631)

  12. Scissors for plant handling (Amazon, any brand)

Software

  1. LI-8x0 v1.02 (LI-COR, Inc., https://www.licor.com/env/support/LI-850/software.html)

  2. R studio (R Core Team, https://www.r-project.org/)

Procedure

  1. Seedling growth

    1. First, sterilize Arabidopsis seeds by adding 100% ethanol to seeds in an Eppendorf tube for 1 min and inverting the tube to mix. Pipette in 1 ml bleach solution (see Recipes). Mix by inversion for 5 min. Remove bleach solution and resuspend seeds in sterile dH2O, mixing for 1 min. Repeat the wash with dH2O three more times and resuspend seeds in sterile 0.01% agar. Spread seeds in a Petri plate containing solid one-half strength MS-phytagel media. Move plates to a dark cold room (4°C) to stratify seeds for 2 days. Place plates under light for 5 days to allow germination to occur. Transfer germinated seedlings to a Falcon 6-well culture plate, with each well containing around 30 ml liquid one-half strength 1/2 MS liquid media (see recipe). Before measurements, allow seedlings to grow (22°C, 12 h light/dark) for an additional 18 days in an orbital shaker (placed inside the plant growth room) at slow speed (Figure 1).



      Figure 1. Seedling growth in 1/2 MS liquid media after 23 days (12 h light/dark)


  2. Respiration measurement preparation

    1. Assemble the light- and airtight custom respiration chamber by attaching the threaded PVC pipe to the quick-connect fittings sealed with the rubber seals. Connect the Balston filter between the sample and the LI-850 in-flow direction using straight unions. The bulkhead connectors go on the ends of tubing that connect to the LI-850 (Figure 2). Check the connection to make sure that there is no air leakage by submerging in water and checking for air bubbles, or gently blowing on the chamber and checking that the CO2 levels in the chamber do not increase. Use Parafilm or petroleum jelly to fix any leaks that may interfere with instrument measurements.



      Figure 2. Custom respiration chamber assembly. (A). Custom respiration chambers comprised of a (1) threaded PVC pipe and (2) threaded cap with (3) quick-connect fittings sealed with rubber seals. (B-G). Close-up view showing the sequential assembly order of the male quick-connect fitting.


    2. Power on the bead bath and set to 23°C and allow time for the temperature to become uniform.

    3. Power on LI-850 and connect to a computer USB port. Open “LI-8x0” software and allow the machine to warm up to an operating temperature of 51°C (approximately 10 min). Choose data logging rate of one reading per second and a tab-delimited .csv file output type.

    4. With the chamber empty, connect it to the LI-850, bury the chamber in the bead bath (Figure 3), type in a filename for the control reading in the LI-850 software, and then press “Start” to log CO2 concentration in the chamber. Start timer and log for 2 min 30 s before pressing “Stop” logging (Figure 4). This should generate a stable CO2 concentration as a check to ensure the equipment is set up correctly (horizontal line on the live time by CO2 graph).



      Figure 3. LI-850 and custom respiration chamber assembly. (A) The LI-850 is connected by USB (1) and data logged to a laptop running the “LI-8x0” software. The chamber (2) is connected by tubing (3) to the LI-850. A Balston filter (4) is fitted between the chamber and the LI-850 in-flow direction using straight unions. Bulkheads (5) go on the ends of tubing that connect to the LI-850. (B) Chamber connected to the LI-850. (C and D) For the respiration measurements, the chamber cap is removed and the sample placed inside. The chamber is then closed and buried in a bead bath (6).



      Figure 4. Shoot respiration data collection and analysis. (A) Connect the LI-850 to a laptop running the “LI-8x0” software. (B) Select instrument default options. (C and D) Select file output readings. (E) Save file and start measurements. (F) Exported LICOR.txt files are then batch processed in R to calculate the rate of CO2 accumulation in the chamber and, therefore, of respiration.


  3. Fresh biomass and dark respiration data collection

    1. Wrap an entire 6-well culture plate containing the growing plants in aluminum foil for 1 h before CO2 flux measurements. For shoot dark respiration, operate in a dark room with a supplemental photosynthetically inactive yellow LED light.

    2. Carefully sever and separate the shoot material from the root material. Record the fresh weight of shoots using a scale before transferring them into a light- and airtight chamber described in Step B1 (see Table S1 for examples of fresh weights recorded).

    3. After recording the fresh weight (Step B1), connect the chamber containing shoot material to the LI-850 immediately. Type in the sample name into the LI-850 software and press “Start” to log CO2 concentration in the chamber. Start timer and record CO2 data for 2 min 30 s before pressing “Stop.” A detectable accumulation of CO2 in the chamber should be observed during this time if the equipment is set up correctly (positive slope on the live time by CO2 graph). As the shoot material has been severed from the whole plant, respiration measurements longer than 10 min are not recommended.

    4. Disconnect the chamber from the LI-850 and use a compressed air duster to remove all plant material from the chamber. Connect the LI-850 to a chamber filled with Drierite for at least one minute between samples to remove any moisture accumulated. Ensure that the LI-850 live CO2 readings have returned to atmospheric levels before connecting the next sample.

    5. Repeat steps 5 and 6 with the remaining samples.

    6. One “.TXT” file containing the raw CO2 concentration measurements over time will be generated per sample. Transfer all generated LI-850 output files to a folder for data analysis.

Data analysis

  1. In R, run the “LI850_respiration.R” code file (see supplemental document) and set the R working directory to a folder containing a copy of the generated LI850 “.TXT” files and a shoot fresh weight file containing all shoot weights “ShootFreshWeight.CSV.”

  2. Optionally, change the dead band value in the code from the default parameter of 60 s to filter out values before the respiration measurement stabilizes. Here, the first 60 s of the 2 min 30 s reading was removed as the LI-850 measurements CO2 flux rate stabilized.

  3. Follow through the code to calculate specific shoot respiration rates (nmol CO2 gfw-1 s-1) in batch for all samples in the folder. Specific shoot respiration rates are calculated using a linear regression of the CO2 flux rate, and then the value is divided by total shoot fresh weight (see Table S1).

  4. Use the forward and back arrows in the plots pane of RStudio to browse through the linear regression graphs for each sample and ensure all are expected with a positive and tight slope.

  5. Save the processed data as a “.CSV” file.

Notes

This protocol is best performed by two people over a period of several hours. In our case, it took 3-4 h to process 10 samples with 5-6 replicates. In our experimental setup, one researcher handled the samples and measured fresh weight using a balance while the other handled the CO2 flux measurements using the LI-850. Additional researchers operating multiple LI-850 machines can increase the sample throughput. This protocol can also be broadly applied to other tissue types, such as plant roots, for measuring respiration rates.

Recipes

  1. 1/2 MS media pH 5.7

    Murashige & Skoog (MS) media with vitamins 2 g

    Sucrose 3 g

    dH2O 800 ml

    Adjust pH to 5.7

    Fill container up to 1 L with dH2O

    Add 8 g of Agar if preparing solid media at this point

    Autoclave at 121°C for 15 min

  2. Bleach solution

    1:1 bleach and dH2O

    0.02% Triton X-100

Acknowledgments

This work was supported by the Noble Research Institute, LLC. This protocol was derived from Fonseca et al. (2020) (doi: 10.1104/pp.20.00950).

Competing interests

The authors declare no financial and non-financial competing interests.

References

  1. Amthor, J. S., Bar-Even, A., Hanson, A. D., Millar, A. H., Stitt, M., Sweetlove, L. J and Tyerman, S. D. (2019). Engineering Strategies to Boost Crop Productivity by Cutting Respiratory Carbon Loss.The Plant Cell 31: 297-314.
  2. Bunce, J. A. (2021). Three new methods indicate that CO2 concentration affects plant respiration in the range relevant to global change. AoB Plants 13(1): plab004.
  3. Condori-Apfata, J. A., Batista-Silva, W., Medeiros, D. B., Vargas, J. R., Valente, L. M. L., Heyneke, E., Pérez-Diaz, J. L., Fernie, A. R., Araújo, W. L and Nunes-Nesi. (2019). The Arabidopsis E1 subunit of the 2-oxoglutarate dehydrogenase complex modulates plant growth and seed production. Plant Mol Biol 101: 183-202.
  4. Fonseca, J. P., Lee, H. K. Boschiero, C., Griffiths, M., Lee, S., Zhao, P. X., York, L and Mysore, K. S. (2020). Iron-sulfur cluster protein NITROGEN FIXATION S-LIKE 1 and its interactor FRATAXIN function in plant immunity. Plant Physiol 184(3): 1532-1548.
  5. Graham, D. (1980). Effects of light on “dark” respiration. In: Davies, D. D. (Ed.). The Biochemistry of Plants. Vol 2. Academic Press, New York, pp 525-579.
  6. Guo, H., Habtamu, A., Seethepalli, A., Dhakal, K., Griffiths, M., Ma, X and York, L.M. (2021). Functional phenomics and genetics of the root economics space in winter wheat using high-throughput phenotyping of respiration and architecture. New Phyt doi: 10.1111/nph.17329.
  7. Holland, B.L., Monk, N.A.M., Clayton, R.H and Osborne, C.P. (2019). A theoretical analysis of how plant growth is limited by carbon allocation strategies and respiration. In Silico Plants 1(1): 1-18.
  8. Li, X., Zhang, G., Sun, B., Zhang, S., Zhang, Y., Liao, Y., Zhou, Y., Xia, X., Shi, K and Yu, J. (2013). Stimulated leaf dark respiration in tomato in an elevated carbon dioxide atmosphere. Sci Rep 3: 3433.
  9. Lill, R. (2009). Function and biogenesis of iron–sulphur proteins. Nature 460(7257): 831-838.
  10. Martin, B., Ort, D.R and Boyer, J.S. (1981). Impairment of photosynthesis by chilling-temperatures in tomato. Plant Physiol 68(2): 329-334.
  11. Tomaz, T., Bagard, M., Pracharoenwattana, I., Lindén, P., Lee, C.P., Carroll, A.J., Ströher, E., Smith, S.M., Gardeström, P and Millar, A.H. (2010). Mitochondrial malate dehydrogenase lowers leaf respiration and alters photorespiration and plant growth in Arabidopsis. Plant Physiol 154(3): 1143-1157.

简介

[摘要]暗呼吸是指在不存在光的叶呼吸的实验措施,做到区分它从所述光合作用过程中发生光呼吸。暗有氧呼吸反应仅发生在线粒体中,并将​​来自细胞质糖酵解和氧气的葡萄糖分子转化为二氧化碳和水,同时生成 ATP 分子。以前的方法通常使用氧传感器来测量Ò xygen耗尽或复杂且昂贵光合作用仪器测量CO 2积聚。在这里,我们提供了一种详细的、循序渐进的方法,通过记录拟南芥芽和根组织的CO 2通量来测量植物的暗呼吸。简而言之,将植物黑暗驯化1小时,切下叶子和根并分别放置在密闭室中,并用标准红外气体分析仪随时间测量CO 2积累。时间序列数据使用 R 脚本处理以产生暗呼吸率,可以通过新鲜或干燥的组织质量进行标准化。目前的方法需要廉价的红外气体分析仪、用于腔室的现成部件和公开可用的数据分析脚本。


[背景]植物通过线粒体呼吸(葡萄糖 + 氧气 = 二氧化碳 + 水)从葡萄糖氧化产生 ATP 形式的能量。为了从固有的光合作用光呼吸分化,暗呼吸测量是由在不存在光的(格雷厄姆,1980) 。线粒体是参与能量生产至关重要的细胞器,氨基酸生物合成,铁-硫(FE-S)簇辅因子的生物合成,以及影响生长和胁迫应答其他代谢途径(利尔,2009) 。我们鉴定了两种线粒体定位的 Fe-S 簇蛋白,NITROGEN FIXATION S-LIKE1 (NFS1)及其相互作用物 FRATAXIN (FH) ,作为植物防御反应的新参与者(Fonseca等,2020)。在当前的手稿中,我们描述了先前研究中使用的方法,通过测量整个芽和根的暗呼吸来测试nfs1和fh突变体以及过表达系对线粒体呼吸的可能影响。这个简单的协议可以使用完全在生长室受控条件下生长的植物来执行。它涉及暗呼吸的通量测量副产物CO 2从在小气密室组织,使用一个LI-850 CO 2 / H 2 O气体分析仪(LICOR)在暗室中与补充光合非活性黄色LED的光。的CO 2对于每个工厂通量是使用软件LI-8X0 V1.02(LICOR)2分钟30秒的时间间隔期间测量的。具体枝条和根呼吸速率小号(纳摩尔CO 2 GFW -1小号-1 )是从原始CO计算出2 ,使用R通量数据(v.3.6.0; https://www.r-project.org/),其中线性回归被用于计算CO 2与流速的在60秒死区集,然后通过总射击鲜重分割。
先前对植物呼吸测量的描述通常包括由独立设备组成的复杂设备,这些设备包括移动空气和使用红外气体分析仪测量 CO 2的独立设备,以及使用质量流量控制器(Martin等人,1981 年;Condori-Apfata等人,2019 年))。最近,比此处使用的气体分析仪贵一个数量级的光合作用仪器已被使用并进行了定制修改(Tomaz等,2010)。作为替代测量CO的产生2 ,其它协议利用氧传感器小号来代替测量ö 2耗尽(李等人,2013年)。相比之下,我们使用更便宜、相对更简单的现成零件实现了相同的目标,这些零件可以由任何工厂实验室组装和使用。此外,我们还公开了用于数据分析的 R 脚本,以确保该方法的可重复性。
该协议对于需要测量暗呼吸作为线粒体丰度和/或活动变化的代表的研究人员非常有用,正如我们在 Fonseca等人所做的那样。(2020 年)。然而,正如光合作用所做的那样,呼吸也被假设为提高代谢效率的潜在目标(Amthor等人,2019 年)。模拟结果强调了将呼吸作为一种未被重视的作物改良途径的机会,在这种情况下,全植物碳分配分析需要考虑叶和根呼吸(Holland等,2020)。实际上,此处概述的类似协议用于测量1,104 株小麦幼苗的根系呼吸,以进行全基因组关联研究(Guo等,2021)。这项工作发现了特定根呼吸的大量遗传变异以及负责该变异的候选基因。Bunce (2021)表明,用于呼吸的CO 2消耗方法与呼吸的总质量减少估计值非常匹配,从而验证了该方法。因此,这种可重复的协议可以对植物生物学的多个领域产生影响,从理解呼吸的分子基础到研究作为育种目标的呼吸。

关键字:NFS1, 暗呼吸, 二氧化碳, 线粒体, 通量


材料和试剂

 
猎鹰6孔透明平底板(Corning,Ç atalog号:353224)
培养皿(15 × 100mm)上(北卡罗来纳州,Ç atalog号:741251)
7.5厘米3内部容积不透明的PVC管,带螺纹的端部(美国塑料公司,1/4" × 6"的表80 CPVC乳头,Ç atalog号码:30207)
具有1/8”螺纹盖孔中钻出的(FNPT尼龙带螺纹的盖,中心美国塑料公司,1/4" Ç :62164 atalog数)
快速连接男性和女性的配件(LI-COR公司,Ç atalog号码:300-07124和300-07125)
Balston滤波器(LI-COR公司,Ç atalog号码:300-01961)
橡胶密封件(LI-COR公司,Ç atalog号码:167-07256)
石蜡膜中号实验室膜(亚马逊,Ç atalog数:PM999)
BEV-A线1/4 '' OD15米管(LI-COR公司,Ç atalog号:8150-250)
快速连接,阴隔板接头(LI-COR公司,Ç atalog号码:300-07126)
快速连接,插头舱壁(LI-COR公司,Ç atalog号码:300-07127)
快速连接直联盟(LI-COR公司,Ç atalog号码:300-03123)
铝箔(亚马逊,任何品牌)
燥石膏(LI-COR公司,Ç atalog号码:622-04299)
拟南芥种子 (ABRC., https://abrc.osu.edu )
含有维生素的Murashige & Skoog(MS)培养基(Grainger,目录号:M70800-50.0)
蔗糖(VWR,目录号:BDH9308-500G)
氢氧化钾(KOH)(VWR,目录号:BDH9262-500G)
的Phytagel(西格玛- Aldricht,Ç atalog号:71010521)
无水乙醇100%(VWR,Ç atalog号:71006012)
的Triton X-100(菲舍尔科学,Ç atalog号:AC327372500)
次氯酸钠8%(次氯酸钠消毒漂白,Ç atalog号:CLO32260)
1 / 2 MS液体培养基 pH 5.7(见配方)
漂白溶液(见配方)
 
设备
 
轨道摇床(THERMOLYNE更大的法案,Ç atalog号:M49235 )
LI-850 CO 2 / H 2 ö分析仪(LI-COR公司Ç atalog编号:)
带有 Windows 10 和 USB 端口的计算机,用于运行 LI-COR 软件
珠浴(实验室护甲,Ç atalog号:74300-714 )
无菌柜(Thermo Scientific,目录号:13-261-221)
高压釜(Steris Amsco Century,目录号:SG-120)
金属珠(实验室护甲,Ç atalog号:42370-004)
计时器/秒表(Big Digit Timer,目录号:ML5004)
压缩空气除尘器(Blow Off Duster,目录号:152-112-226)
实验室天平(梅特勒托利多,目录号:ME103TE)
用于植物处理的弯曲尖端钳(Epredia,目录号:1631)
植物处理剪刀(亚马逊,任何品牌)
 
软件
 
LI-8x0 v1.02(LI-COR, Inc. ,https: //www.licor.com/env/support/LI-850/software.html )
R工作室(R 核心团队,https://www.r-project.org/)


程序
 
幼苗生长
首先,灭菌拟南芥通过在加入100%乙醇种子种子Ë ppendorf管1分钟,翻转荷兰国际集团的管以混合。移液管在1米升漂白溶液(见ř ecipes) 。颠倒混合 5 分钟。除去在无菌卫生署漂白溶液和重悬种子2 O,混合荷兰国际集团1分钟。重复的WA与卫生署SH 2 ö三次以上,重悬种子在无菌0.01%琼脂中。在含有固体二分之一强度 MS-phytagel 培养基的培养皿中传播种子。将板移到黑暗的冷室 (4°C) 中,将种子分层 2 天。将板置于光照下 5 天以允许发芽。转印发芽苗猎鹰6孔培养板,每孔含有约30米升液体的二分之一强度1/2 MS液体培养基(见配方)。乙EFORE测量,一个llow幼苗生长(22℃,12小时光照/黑暗),用于在SLO轨道摇床(放置在植物生长室的内部)的附加18天瓦特速度(图1)。
 
 
图1 。23 天(12 小时光/暗)后,幼苗在 1/2 MS 液体培养基中的生长
 
呼吸测量准备
1.通过将带螺纹的 PVC 管连接到用橡胶密封件密封的快速连接配件,组装轻巧且密封的定制呼吸室。使用直管接头在样品和 LI-850 流入方向之间连接Balston 过滤器。隔板连接器位于连接 LI-850 的管道末端(图 2)。检查连接,以确保有是没什么漏气水ubmerging和检查荷兰国际集团的气泡,或轻轻地吹在室内,检查荷兰国际集团的CO 2的室水平不增加。使用封口膜或凡士林修复可能干扰仪器测量的任何泄漏。                    
 
 
图 2. 定制呼吸室组件。(一)。Ç由一个(1)ustom呼吸室螺纹PVC管和(2)的螺纹与(3)的橡胶密封件密封快速连接管接头帽。(B - G) 。特写视图显示了公快速连接接头的顺序组装顺序。
 
2.开启珠浴并设置为 23 °C ,让温度变得均匀。      
3.打开 LI-850 并连接到计算机 USB 端口。打开“LI-8x0”软件,让机器预热到 51 °C的工作温度(大约 10 分钟)。选择每秒一次读数的数据记录速率和制表符分隔的.csv 文件输出类型。      
4.随着该室排空,将其连接到LI-850,掩埋室在胎圈浴(图3)中,为在LI-850软件控制读取,然后按“启动”到日志文件名腔室中的CO 2浓度。在按“停止”记录之前启动计时器并记录 2 分 30 秒(图 4)。个是应该生成稳定的CO 2浓度作为检查,以确保设备正确设置(由CO在实时时间水平线2图)。      
 
 
 
图 3. LI-850 和定制呼吸室组件。(A) LI-850通过 USB (1)连接,数据记录到运行“LI-8x0”软件的笔记本电脑。腔室(2)通过管道(3)连接到 LI-850。Balston 过滤器(4)使用直管接头安装在腔室和 LI-850 流入方向之间。隔板(5)位于连接 LI-850 的管道末端。(B)Ç hamber连接到LI-850 。(C和d)对于呼吸作用measuremen TS,T他室盖被移除并且样品置于其内。该腔室被再关闭和埋设在胎圈浴(6) 。
 
 
图 4.射击呼吸数据收集和分析。(A) 将LI-850连接到运行“LI-8x0”软件的笔记本电脑。(B)小号选仪器默认选项。(C 和 D) S选择文件输出读数。(E)š AVE文件,并开始测量。(F)导出LICOR.txt文件然后批次中的R处理以计算CO的速率2积累在腔室和,因此,的呼吸。
 
新鲜生物量和暗呼吸数据收集
在CO 2通量测量之前,将含有生长植物的整个 6 孔培养板包裹在铝箔中 1 小时。对于拍摄暗呼吸,请在带有补充光合作用非活动黄色 LED 灯的暗室中操作。
小心地将芽材料与根材料分开。记录鲜重的使用分的转印之前枝条米到光描述的和气密腔室在步骤B1 (见表S1为例如小号的鲜重记录小号)。
记录鲜重(步骤B1)后,连接含有枝条材料到LI-850的腔室立即。键入的样品名称到的LI-850软件,然后按“启动”登录CO 2在腔室中的浓度。在按下“停止”之前启动计时器并记录CO 2数据 2 分 30 秒。”如果设备设置正确,则在此期间应观察到可检测到的 CO 2积聚在腔室中(CO 2图的实时时间呈正斜率)。由于芽材料已从整株植物中分离出来,因此不建议呼吸测量时间超过 10 分钟。
将腔室与 LI-850 断开连接,并使用压缩空气除尘器从腔室中清除所有植物材料。850 LI-连接到填充有燥石膏样品间至少一分钟,以除去一个腔室的任何湿气accumulat版。保证是在LI-850现场CO 2的读数已经下一个样品连接之前恢复到大气水平。
对剩余的样品重复步骤 5 和 6。
每个样本将生成一个包含随时间变化的原始 CO 2浓度测量结果的“.TXT”文件。将所有生成的 LI-850 输出文件传输到一个文件夹进行数据分析。
 
数据分析
 
在 R 中,运行“ LI850_respiration.R ”代码文件(参见补充文档)并将 R 工作目录设置为包含生成的 LI850“.TXT”文件副本和包含所有芽重“ShootFreshWeight”的芽鲜重文件的文件夹.CSV 。”
或者,将代码中的死区值从 60 s 的默认参数更改为在呼吸测量稳定之前过滤掉值。在这里,2 分 30 秒读数的前 60 秒被删除,因为 LI-850 测量 CO 2通量率稳定。
按照代码计算文件夹中所有样本的特定芽呼吸速率 (nmol CO 2 gfw -1 s -1 )。枝条特异性呼吸速率小号是使用CO的线性回归计算2通量速率,并然后将该值是由总射击鲜重划分(参照表S1)。
使用RStudio绘图窗格中的向前和向后箭头浏览每个样本的线性回归图,并确保所有样本都具有正斜率。
将处理后的数据另存为“.CSV”文件。
 
笔记
 
该协议最好由两个人在几个小时内执行。在我们的案例中,处理10 个样品 5-6 次重复需要3-4小时。在我们的实验设置中,一位研究人员使用天平处理样品并测量鲜重,而另一位研究人员使用 LI-850处理 CO 2通量测量。操作多台 LI-850 机器的其他研究人员可以增加样品吞吐量。该协议还可以广泛地应用于其它组织类型,如植物的根部,用于测量电荷兰国际集团呼吸速率。
 
食谱
 
1 / 2 MS 介质 pH 5.7
Murashige & Skoog (MS) 培养基,含维生素2 克
蔗糖3 克
卫生署2 ö800米升
将 pH 值调整为 5.7
用 dH 2 O填充容器至 1 L
如果此时准备固体培养基,则添加 8克琼脂
121°C 高压灭菌 15 分钟
漂白液
1:1 漂白剂和 dH 2 O
0.02%Ť瑞通X-100
 
致谢
 
这项工作得到了 Noble Research Institute, LLC 的支持。该协议源自 Fonseca等人。(2020 年)(doi:10.1104/pp.20.00950)。
 
利益争夺
 
作者声明没有财务和非财务竞争利益。
参考
 
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Fonseca, J. P., Griffiths, M., York, L. M. and Mysore, K. S. (2021). Dark Respiration Measurement from Arabidopsis Shoots. Bio-protocol 11(19): e4181. DOI: 10.21769/BioProtoc.4181.
  2. Fonseca, J. P., Lee, H. K. Boschiero, C., Griffiths, M., Lee, S., Zhao, P. X., York, L and Mysore, K. S. (2020). Iron-sulfur cluster protein NITROGEN FIXATION S-LIKE 1 and its interactor FRATAXIN function in plant immunity. Plant Physiol 184(3): 1532-1548.
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