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Determination of the Effects of Local and Systemic Iron Excess on Lateral Root Initiation in Arabidopsis thaliana
局部和系统性铁过量对拟南芥侧根起始影响的测定   

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Plant Physiology
Dec 2015

Abstract

Root system architecture depends on nutrient availability. A symptom of iron (Fe) toxicity in plants is stunted root growth, yet little is known about the effects of excess Fe on lateral root (LR) development. To better understand how nutrient signals are integrated into root developmental programs, we investigated the morphological response of Arabidopsis thaliana root systems to Fe by testing homogeneous supply and localized Fe supply treatment.

Keywords: Arabidopsis (拟南芥), Fe toxicity (铁毒性), Lateral root (侧根), Localized iron supply (局部铁供应), Homogeneous iron supply (均质铁供应)

Background

A localized supply of nutrients regulates lateral root elongation and/or lateral root density in Arabidopsis thaliana. Lateral root formation is affected by nutrients at different developmental stages (e.g., initiation versus elongation) and in a nutrient-specific manner (Li et al., 2011; Giehl et al., 2012). Iron toxicity as a syndrome in plants is typically associated with an excessive amount of the ferrous form (the Fe2+ ion) in the soil (Vigani, 2012). Iron toxicity symptoms vary with cultivars and are characterized by a reddish-brown, purple bronzing, yellow, or orange discoloration of the lower leaves and a stunted root growth. The presence of the Fe2+ ion is increased by low pH and anoxic conditions, and there is an increasing presence in vertically lower strata (Becker and Asch, 2005; Li et al., 2016). We describe a detailed pipeline used for localized iron supply in Arabidopsis grown in vitro which we validated in Arabidopsis (Li et al., 2015a; 2015b and 2016).

Materials and Reagents

  1. Eppendorf tubes (1.5 ml)
  2. Sterile tips
  3. Plastic wrap (Bemis, catalog number: PM996 )
  4. 130 x 130 x 12 mm square plastic plates (self-made, see Figure 1)
  5. 130 x 13 x 2 mm glass strip (self-made, see Figure 1)


    Figure 1. Square plastic plates used for treatment

  6. Arabidopsis thaliana seeds (DR5:GUS lines, Col-0 background)
  7. Distilled water
  8. Ethanol (Sinopharm Chemical Reagent, catalog number: 80176961 )
  9. Sodium hypochlorite (NaClO) (Sinopharm Chemical Reagent, catalog number: 80010428 )
  10. Sodium dodecyl sulfate, sodium salt (SDS) (Sinopharm Chemical Reagent, catalog number: 30166428 )
  11. Potassium phosphate monobasic (KH2PO4) (Sinopharm Chemical Reagent, catalog number: 10017618 )
  12. Sodium nitrate (NaNO3) (Sinopharm Chemical Reagent, catalog number: 10019918 )
  13. Magnesium sulfate (MgSO4) (Sinopharm Chemical Reagent, catalog number: 10013018 )
  14. Calcium chloride (CaCl2) (Sinopharm Chemical Reagent, catalog number: 20011160 )
  15. Ferrous sulfate (FeSO4·7H2O) (Sinopharm Chemical Reagent, catalog number: 10012116 )
  16. Ethylene diamine tetraacetic acid (EDTA) (Sinopharm Chemical Reagent, catalog number: 10009717 )
  17. Boric acid (H3BO3) (Sinopharm Chemical Reagent, catalog number: 10004818 )
  18. Manganese sulfate (MnSO4) (Sinopharm Chemical Reagent, catalog number: LB2208102 )
  19. Zinc chloride (ZnCl2) (Sinopharm Chemical Reagent, catalog number: 10023828 )
  20. Copper sulfate (CuSO4) (Sinopharm Chemical Reagent, catalog number: 10008216 )
  21. Sodium molybdate (Na2MoO4) (Sinopharm Chemical Reagent, catalog number: 10019818 )
  22. Sucrose (Sinopharm Chemical Reagent, catalog number: 10021418 )
  23. MES hydrate (Sigma-Aldrich, catalog number: M8250 )
  24. Agar-agar (Sigma-Aldrich, catalog number: A7002 )
  25. Seed sterilization solution (see Recipes)
  26. Normal growth medium (see Recipes)
  27. Fe-supplemented medium and the control medium (see Recipes)
  28. Control medium (see Recipes)

Equipment

  1. Glass bottles (Schott Duran glass bottle, 500 ml capacity or higher)
  2. Incubation chamber (23 ± 1 °C, under fluorescent lamps at 100 μmol/m2/sec, 16-h-light/8-h-dark)
  3. pH meter (Mettler-Toledo International, model: FE20K )
  4. Autoclave (TOMY DIGITAL BIOLOGY, model: SX-500 )
  5. Refrigerator (Haier, model: BCD-648WDBE )
  6. Flow hood (Suzhou Antai Air-tech, model: SW-CJ-1F(D) )
  7. Vortex
  8. Tweezers

Procedure

  1. The following media are required (see Recipes section): (a) normal growth medium, for seed germination, (b) control medium which contains 50 μM Fe-EDTA and (c) Fe-supplemented medium which contains 350 μM Fe-EDTA.
  2. Seed sterilization and plating of seeds.
    1. Put 5 mg (200-300 seeds) of Arabidopsis seeds into an Eppendorf tube and add 750 µl sterilization solution (see Recipes).
    2. Vortex for 1 min at room temperature.
    3. Decant the sterilization solution (with sterile tips) under a flow hood and add 750 µl sterile water to wash the seeds.
    4. Vortex briefly and decant the water.
    5. Repeat the wash step 5 times.
    6. Stratify the seeds in 750 µl sterile water at 4 °C for 2 days in the dark.
    7. Sow seeds on sterile normal growth medium plates. Next close and seal the plates with plastic wrap.
    8. Incubate plates in a vertical position in a growth chamber at 23 °C with a 16-h-light/8-h-dark cycle (150 μmol m-2 sec-1).
    9. Grow seedlings for 5-7 days (with a primary root length of about 2 cm).
  3. Whole root and localized root supply of excess Fe.
    1. Prepare the plates (see the Figure 2), and at least three replicates (plates) are needed per treatment.


      Figure 2. Prepare the plates for treatment. The glass strip and tweezers are autoclaved. Place the glass bars in the Petri dishes with tweezers and the glass bar must be fixed tightly in the air gap.

    2. Preparation of agar media for uniform and localized Fe treatments. The agar media is firstly autoclaved for 20 min at 121 °C on the liquid cycle. The Fe-supplemented medium can be set up as follows. For (I) uniform-Fe supplemented medium, spread the treatment media evenly (Figure 3C). For (II) ‘root tip-Fe supplemented medium’, use segmented agar plates that are separated into upper and bottom parts with a 3-mm air gap that is made with 3 mm-wide movable glass strips. Pour the control medium into the upper part and Fe-supplemented medium into the bottom part (Figure 3D). For (III) ‘shoot-Fe supplemented medium’, pour control medium into the bottom part and pour Fe-supplemented medium into the upper part (Figure 3E). The composition of the Fe-supplemented medium (350 μM Fe-EDTA) and control medium (50 μM Fe-EDTA) is similar to the normal growth medium but the pH is 5.3.


      Figure 3. Experimental setup for preparing control and Fe-supplemented medium. The control medium appears light gray; Fe-supplemented medium appears dark gray.

    3. For Fe treatment of the seedlings. Germinate the seedlings (DR5:GUS lines) for 5 to 7 days and then transfer them to Fe-supplemented media with forceps (gently stir up the seedlings but do not pick them up) (Figure 4A). For ‘root tip-supplied’ plants, arrange so that only the primary root tip of the seedlings (~2 mm) is in contact with the bottom of the medium are referred to as ‘root tip-supplied Fe’ plants (Figure 4B). For ‘shoot-supplied Fe’ plants, arrange so that the shoot and mature primary root zone of the seedlings are in contact with the upper parts of the medium. The duration of the treatment is 5 days. All initiation events, including emerged LRs and LRP, are quantified using DR5:GUS lines (Figures 4C and 4D).


      Figure 4. Effect of local supply of Fe on LR initiation in Arabidopsis. A. Schematic diagram of the experimental setup for applying excess Fe to the whole root versus the root tip and the shoot-mature root continuum. White sections indicate the basal growth medium with the control medium, and gray sections indicate the Fe-supplemented medium (350 μM Fe-EDTA). B. A picture of seedlings after transfer to the final media; C. Images of lateral roots observed under the microscope (Zhang et al., 1999); D. The effect of locally supplied Fe on LR initiation at 5 day. Values are means of 6 plants ± SE. Ctrl, Control medium.

Data analysis

After treatment count the number of lateral root initiation events under a dissecting microscope. The developmental stage of each lateral root primordia (LRP) is classified according to Zhang et al. (1999) into the following stages: stage A, up to three cell layers; stage B, unemerged, more than three cell layers; stage C, emerged LRs, less than 0.5 mm in length; and stage D, LRs, greater than 0.5 mm. The emerged but not activated LRP are still referred to as LRP, and only mature LRs (exceeding 0.5 mm in length) are denoted as LRs (Figure 4C). The number of lateral root initiation events is stage A + B + C + D (Figure 4D).

Notes

  1. From the seed sterilization step, all the operations must be performed at a clean bench.
  2. For the step of placing the glass bar in the Petri dishes: Firstly, the glass bar and forceps must be sterile; Secondly, the glass bar must be placed in the groove (air gap) of the Petri dishes using forceps. All the operations must be performed at a clean bench.
  3. In Figures 3D and 3E, before moving on to step 3, you have to wait for the agar from step 2 to solidify in the upper part. Similarly, before moving on to step 4, wait for the agar from step 3 to solidify in the bottom part.

Recipes

  1. Seed sterilization solution
    75% (v/v) ethanol
    10% (v/v) NaClO
    0.1% (w/v) SDS
  2. Normal growth medium


  3. Fe-supplemented medium
    Normal growth medium supplemented with 350 μM Fe-EDTA
    Adjust pH to 5.3 with 1 N NaOH
  4. Control medium
    Normal growth medium supplemented with 50 μM Fe-EDTA
    Adjust pH to 5.3 with 1 N NaOH

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31430095) and the Chinese Academy of Sciences Innovation Program (CAS ISSASIP1604). This protocol was adapted from Li et al., 2015a.

References

  1. Becker, M. and Asch, F. (2005). Iron toxicity in rice-conditions and management concepts. J Plant Nutr Soil Sci 168: 558-573.
  2. Giehl, R. F., Lima, J. E., von Wiren, N. (2012). Localized iron supply triggers lateral root elongation in Arabidopsis by altering the AUX1-mediated auxin distribution. Plant Cell 24(1): 33-49
  3. Li, B., Li, Q., Su, Y., Chen, H., Xiong, L., Mi, G., Kronzucker, H. J. and Shi, W. (2011). Shoot-supplied ammonium targets the root auxin influx carrier AUX1 and inhibits lateral root emergence in Arabidopsis. Plant Cell Environ 34(6): 933-946.
  4. Li, G., Kronzucker, H. J. and Shi, W. (2016). The response of the root apex in plant adaptation to iron heterogeneity in soil. Front Plant Sci 7: 344.
  5. Li, G., Song, H., Li, B., Kronzucker, H. J. and Shi, W. (2015a). Auxin resistant1 and PIN-FORMED2 protect lateral root formation in Arabidopsis under iron stress. Plant Physiol 169(4): 2608-2623.
  6. Li, G., Xu, W., Kronzucker, H. J. and Shi, W. M. (2015b). Ethylene is critical to the maintenance of primary root growth and Fe homeostasis under Fe stress in Arabidopsis. J Exp Bot 66, 2041-2054.
  7. Vigani, G. (2012). Discovering the role of mitochondria in the iron deficiency-induced metabolic responses of plants. J Plant Physiol 169(1): 1-11.
  8. Zhang, H., Jennings, A., Barlow, P. W. and Forde, B. G. (1999). Dual pathways for regulation of root branching by nitrate. Proc Natl Acad Sci U S A 96(11): 6529-6534.

简介

根系统结构取决于营养供应。 植物中铁(Fe)毒性的症状是根系生长发育迟缓,但是对于过量的Fe对侧根(LR)发育的影响知之甚少。 为了更好地了解营养信号如何整合到根发育方案中,我们通过测试均匀供应和局部Fe供应处理,研究了拟南芥根系对Fe的形态反应。
【背景】营养素的局部供应调节拟南芥中的侧根伸长和/或侧根密度。侧根形成受不同发育阶段(例如,起始与延长)和营养特异性方式的营养素的影响(Li et al。,2011; Giehl et al。,2012)。作为植物综合征的铁毒性通常与土壤中过量的铁(Fe(OH)2 +离子)相关联(Vigani,2012)。铁毒性症状因栽培品种而异,其特征在于下部叶子呈红褐色,紫色,黄色或橙色变色,发育不良。通过低pH和缺氧条件,Fe 2 + 离子的存在增加,并且在垂直较低的层中存在增加的存在(Becker和Asch,2005; Li et al。 em>,2016)。我们描述了在拟南芥中验证的体外生长的拟南芥中用于局部铁供应的详细管道(eta。 ,2015a; 2015b和2016)。

关键字:拟南芥, 铁毒性, 侧根, 局部铁供应, 均质铁供应

材料和试剂

  1. Eppendorf管(1.5毫升)
  2. 无菌提示
  3. 塑料包装(Bemis,目录号:PM996)
  4. 130 x 130 x 12 mm方形塑料板(自制,见图1)
  5. 130 x 13 x 2 mm玻璃条(自制,见图1)


    图1.用于治疗的方形塑料板

  6. 拟南芥种子(DR5:GUS 线,Col-0背景)
  7. 蒸馏水
  8. 乙醇(国药化学试剂,目录号:80176961)
  9. 次氯酸钠(NaClO)(国药化学试剂,目录号:80010428)
  10. 十二烷基硫酸钠,钠盐(SDS)(国药化学试剂,目录号:30166428)
  11. 磷酸二氢钾(KH 2 PO 4)(国药化学试剂,目录号:10017618)
  12. 硝酸钠(NaNO 3)(国药化学试剂,目录号:10019918)
  13. 硫酸镁(MgSO 4)(国药化学试剂,目录号:10013018)
  14. 氯化钙(CaCl 2)(国药化学试剂,目录号:20011160)
  15. 硫酸亚铁(FeSO 4·7H 2 O)(国药化学试剂,目录号:10012116)
  16. 戊二胺四乙酸(EDTA)(国药化学试剂,目录号:10009717)
  17. 硼酸(H 3 3 BO 3)(国药化学试剂,目录号:10004818)
  18. 硫酸锰(MnSO 4)(国药化学试剂,目录号:LB2208102)
  19. 氯化锌(ZnCl 2)(国药化学试剂,目录号:10023828)
  20. 硫酸铜(CuSO 4)(国药化学试剂,目录号:10008216)
  21. 钼酸钠(Na 2 MoO 4)(国药化学试剂,目录号:10019818)
  22. 蔗糖(国药化学试剂,目录号:10021418)
  23. MES水合物(Sigma-Aldrich,目录号:M8250)
  24. 琼脂(Sigma-Aldrich,目录号:A7002)
  25. 种子灭菌方案(见食谱)
  26. 正常生长培养基(参见食谱)
  27. Fe补充培养基和对照培养基(参见食谱)
  28. 控制介质(见配方)

设备

  1. 玻璃瓶(Schott Duran玻璃瓶,500毫升以上容量)/ /
  2. 孵育室(23±1℃,荧光灯以100μmol/ m 2 /秒以下,16小时/ 8小时黑暗)
  3. pH计(Mettler-Toledo International,型号:FE20K)
  4. 高压灭菌器(TOMY DIGITAL BIOLOGY,型号:SX-500)
  5. 冰箱(海尔,型号:BCD-648WDBE)
  6. 流量罩(苏州安泰空气科技,型号:SW-CJ-1F(D))
  7. 漩涡
  8. 镊子

程序

  1. 需要以下介质(参见食谱部分):(a)正常生长培养基,用于种子发芽,(b)含有50μMFe-EDTA的对照培养基和(c)含有350μMFe-EDTA的Fe补充培养基。
  2. 种子灭菌和种子电镀。
    1. 将5mg(200-300粒种子)的拟南芥种子放入Eppendorf管中,加入750μl灭菌溶液(参见食谱)。
    2. 在室温下涡旋1分钟。
    3. 在流动罩下倒置灭菌溶液(无菌提示),并加入750μl无菌水以洗涤种子。
    4. 短暂旋涡并滗出水。
    5. 重复洗涤步骤5次。








    6. 在无菌正常生长培养基平板上播种种子。接下来关闭并用塑料包装密封板。
    7. 在23℃的生长室中以16小时/ 8小时黑暗的循环(150μmol/平方米以上)向上孵育板垂直位置。 >)。
    8. 种植幼苗5-7天(主根长约2厘米)。
  3. 全根和局部根部供应过量的铁。
    1. 准备板(参见图2),每次处理需要至少三次复制(平板)。


      图2.准备待处理的板。将玻璃条和镊子高压灭菌。将玻璃棒放在培养皿中,用镊子,玻璃棒必须紧紧地固定在气隙中。

    2. 制备用于均匀和局部Fe处理的琼脂培养基。琼脂培养基首先在液体循环121℃下高压灭菌20分钟。 Fe补充介质可以设置如下。对于(I)均匀的Fe补充培养基,将处理介质均匀铺展(图3C)。对于(II)'根尖 - 补充Fe的培养基',使用分割成上部和底部的分段琼脂板,其间距为3毫米,气隙为3毫米宽的可移动玻璃条。将控制介质倒入上部,将Fe补充介质倒入底部(图3D)。对于(III)“补充培养基的培养基”,将控制培养基倒入底部,并将Fe补充培养基倒入上部(图3E)。 Fe补充培养基(350μMFe-EDTA)和对照培养基(50μMFe-EDTA)的组成与正常生长培养基相似,但pH为5.3。


      图3.制备对照和Fe补充培养基的实验装置。 控制介质显示为浅灰色;补铁介质呈灰暗色。

    3. 对于Fe处理幼苗。将幼苗(“DR5:GUS”系列)发芽5至7天,然后用镊子将其转移到补充Fe的培养基中(轻轻搅拌幼苗,但不要接种)(图4A)。对于“根尖提供的”植物,安排使得只有幼苗的主要根尖(〜2mm)与培养基的底部接触被称为“根尖提供的Fe”植物(图4B) 。对于“笋提供的Fe”植物,布置使得幼苗的芽和成熟主根区与培养基的上部接触。治疗的持续时间为5天。所有启动事件,包括出现的LR和LRP都使用DR5:GUS 行进行量化(图4C和4D)。


      图4.在拟南芥中局部供应Fe对LR起始的影响 A.将过量Fe施用于根部与根尖的实验装置的示意图芽成熟根连续体。白色部分表示具有对照培养基的基础生长培养基,灰色部分表示Fe补充培养基(350μMFe-EDTA)。 B.转移到最终媒体后的幼苗图片C.在显微镜下观察到的侧根的图像(Zhang等人,1999); D.当地供应的铁对LR启动的影响在5天。价值是6植物±SE的手段。 Ctrl,控制媒体。

数据分析

治疗后,在解剖显微镜下计算侧根发生事件的数量。每个侧根原基(LRP)的发育阶段根据Zhang等人(1999)分为以下阶段:阶段A,最多三个细胞层;阶段B,未通过,超过三个细胞层;阶段C,出现LR,长度小于0.5毫米;和阶段D,LRs大于0.5毫米。出现但未激活的LRP仍被称为LRP,只有成熟的LR(长度超过0.5mm)被表示为LR(图4C)。侧根发生事件的数量是阶段A + B + C + D(图4D)。

笔记

  1. 从种子灭菌步骤开始,所有的操作都必须在干净的工作台上进行
  2. 将玻璃棒放在培养皿中的步骤:首先,玻璃棒和镊子必须是无菌的;其次,玻璃棒必须使用镊子放置在培养皿的凹槽(气隙)中。所有操作必须在干净的工作台上进行。
  3. 在图3D和3E中,在转到步骤3之前,您必须等待第2步的琼脂在上部固化。类似地,在转到步骤4之前,等待步骤3中的琼脂在底部固化。

食谱

  1. 种子灭菌解决方案
    75%(v / v)乙醇 10%(v / v)NaClO
    0.1%(w / v)SDS
  2. 正常生长培养基


  3. Fe补充培养基
    补充有350μMFe-EDTA的正常生长培养基 用1N NaOH调节pH至5.3
  4. 控制媒介
    补充50μMFe-EDTA的正常生长培养基 用1N NaOH调节pH至5.3

致谢

这项工作得到了国家自然科学基金(31430095)和中国科学院创新计划(CAS ISSASIP1604)的支持。该协议由Li等人,2015a修改。

参考

  1. Becker,M.和Asch,F。(2005)。水稻条件和管理概念中的铁毒性。植物营养土壤科学 168:558-573。
  2. Giehl,RF,Lima,JE,von Wiren,N。(2012)。  通过改变AUX1介导的生长素分布,局部铁供应触发拟南芥的侧根伸长。植物细胞 24(1):33-49
  3. Li,B.,Li,Q.,Su,Y.,Chen,H.,Xiong,L.,Mi,G.,Kronzucker,HJ和Shi,W(2011)。&lt; a class = -insertfile“href =”http://www.ncbi.nlm.nih.gov/pubmed/21342208“target =”_ blank“>拍摄提供的铵靶向根生长素流入载体AUX1并抑制<拟南芥。植物细胞环境34(6):933-946。
  4. Li,G.,Kronzucker,HJ和Shi,W.(2016)。根尖在植物适应土壤中铁异质性的反应。前植物科学 7:344.
  5. Li,G.,Song,H.,Li,B.,Kronzucker,HJ和Shi,W.(2015a)。&nbsp; 植物生理学 > 169(4):2608-2623。
  6. Li,G.,Xu,W.,Kronzucker,HJ and Shi,WM(2015b)。&lt; a class =“ke-insertfile”href =“http://jxb.oxfordjournals.org/content/early/2015 /02/22/jxb.erv005“target =”_ blank“>乙烯对于在拟南芥中Fe胁迫下维持初级根生长和Fe稳态至关重要。 Bot 66,2041-2054。
  7. Vigani,G。(2012)。&nbsp; 发现作用线粒体缺铁诱导的植物代谢反应。植物生理学169(1):1-11。
  8. Zhang,H.,Jennings,A.,Barlow,PW和Forde,BG(1999)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed / 10339622“target =”_ blank“>由硝酸盐调节根分支的双重途径。美国Proc Natl Acad Sci USA 96(11):6529-6534。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Li, G., Zhang, L. and Shi, W. (2017). Determination of the Effects of Local and Systemic Iron Excess on Lateral Root Initiation in Arabidopsis thaliana. Bio-protocol 7(13): e2387. DOI: 10.21769/BioProtoc.2387.
  2. Li, G., Song, H., Li, B., Kronzucker, H. J. and Shi, W. (2015). Auxin Resistant1 and PIN-FORMED2 Protect Lateral Root Formation in Arabidopsis under Iron Stress. Plant Physiol 169(4): 2608-2623.
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