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Protocol for Increasing Carotenoid Levels in the Roots of Citrus Plants
提高柑橘植物根中类胡萝卜素含量的实验方案   

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Abstract

Carotenoids in plants play several key functions such as acting as light-harvesters, antioxidants (Lado et al., 2016) or being precursors of strigolactones, abscisic acid, volatiles and other signaling compounds (Arbona et al., 2013). Although those functions are well-known in light-exposed tissues, information in belowground organs is limited because of reduced abundance of these pigments. In order to better understand the role of carotenoids in roots, we developed a methodology to increase the abundance of these pigments in underground tissues. We took advantage of the fact that citrus roots exposed to light develop pigmentation in order to increase the carotenoid content. Therefore, here we describe a simple method to increase carotenoids in citrus roots.

Keywords: Abscisic acid(脱落酸), Phytohormones(植物激素), Growth chamber(生长室), in vitro culture(体外培养), Root detachment(根脱离), Seed germination(种子萌发)

Background

Carotenoid abundance in roots is quite limited and, therefore, understanding the role of these compounds becomes difficult. Exposure of roots to light is a simple, fast and useful tool to increase carotenoid levels in these tissues, especially when compared to other genomic approaches such as overexpressing some key genes of the carotenoid biosynthetic pathway (Cao et al., 2015).

Materials and Reagents

  1. Pirex® culture tubes 25 x 150 mm
  2. Disposable syringes (25 ml)
  3. 1.5 ml Eppendorf tubes
  4. Citrus seeds (e.g., from a commercial rootstock)
  5. Murashige and Skoog (MS) medium (4,302.09 mg L−1) (Duchefa Biochemie, catalog number: M0221 )
  6. Sucrose (30 g L−1) (any supplier)
  7. Sterile MiliQ-water
  8. Agar (European Bacteriological Agar) (Conda, Pronadisa, catalog number: 1800 )
  9. Tween® 20 (0.1% v/v) (Sigma-Aldrich, catalog number: P2287 )
  10. Myo-inositol (100 mg L−1) (Duchefa Biochemie, catalog number: I0609 )
  11. Pyridoxine-HCl (1.0 mg L−1) (Duchefa Biochemie, catalog number: P0612 )
  12. Thiamine-HCl (0.2 mg L−1) (Duchefa Biochemie, catalog number: T0614 )
  13. Nicotinic acid (0.5 mg L−1) (PANREAC QUÍMICA, catalog number: A0963 )
  14. Glycine (0.2 mg L−1) (PANREAC QUÍMICA, catalog number: 141340 )
  15. Mix A (see Recipes)
  16. Sodium hypochlorite solution (50.0%, v/v) (see Recipes)
  17. NaOH (0.1 N) (any supplier) (see Recipes)

Equipment

  1. Pirex® media solution bottles
  2. Autoclave (Raypa, model: Steam Sterilizer AES-75 )
  3. Laminar flow hood (Bioquell, ASTEC MICROFLOW, model: Microflow Laminar Flow Workstation M50546 )
  4. Hooked tweezers
  5. Growth chamber (Snijders Scientific, model: Economic Lux Climate Chamber )
  6. Long-handled scissors with curved tip
  7. pH meter (HACH LANGE SPAIN, model: pH meter Basic 20 )

Procedure

  1. Prepare growing medium
    1. Dissolve 4.3 g L-1 of MS, 1.0 ml of mix A (see Recipes) and 30 g L-1 of sucrose in distilled water.
    2. Adjust the pH of the solution to 5.7 ± 0.1 with NaOH (0.1 N).
    3. Distribute the solution in bottles for autoclaving.
    4. Autoclave the solution at 105 °C for 5 min.
    5. Dissolve 9.0 g L-1 of agar to solidify the medium.
    6. Even with the warm medium and by means of syringes, dispense 25 ml of the solution into the culture tubes and cover it.
    7. Autoclave the racks containing tubes for 15 min at 115 °C.
    8. Let it cool down and store at room temperature.

  2. Plant material preparation
    1. Remove the coats of the seeds to increase and homogenize seed germination. Avoid damaging the seeds; especially pay attention not to injure the embryo (Figure 1). Disinfect the seeds by putting them into a sodium hypochlorite solution (50.0%, v/v) plus 0.1% (v/v) Tween® 20 for 10 min.
    2. Rinse the seeds three times with sterile distilled water in order to remove hypochlorite solution.
    3. Store the seeds in a sterile laminar flow hood preventing dehydration.


      Figure 1. Example of seeds coat removal for increasing the rate and homogenization of germination

  3. Germination and growing conditions
    1. Working in a sterile laminar flow hood and by means of a sterile tweezer, place one seed per tube in the solidified growing medium and cover the tubes after sown (Figure 2A).
    2. Once seeds are sown place racks into a growing chamber for two weeks maintained at 25 °C and darkness to promote germination (Figure 2B).
    3. After two weeks, transfer the etiolated seedlings (of about 5 cm in large at that time) to a lighted growth chamber. Set photoperiod conditions of 16:8 h (light:dark), temperature of 25 °C and a minimum radiation of 150 µmol m-2 s-1 photosynthetically active radiation (PAR) (Figure 2C).
    4. After three weeks under light conditions when plants have grown and turned to green coloration, shoot needs to be removed. In the sterile laminar flow hood plants are detached from shoot by means of long-handled scissors (Figure 3). Do this under sterile conditions in the laminar flow hood. Shoots are removed by cutting about 5 mm below of the root/shoot junction, keeping the remaining root inside the medium to avoid future shoot regrowth (Figure 2D).
    5. Tubes containing root tissues are maintained for at least two days prior to experiments are performed to exclude any side-effect of wounding (Figure 4).


      Figure 2. Example of seedling development under in vitro condition. A. Sown seeds. B. Etiolated seedlings growing under dark conditions. C. Seeds turned green after 3 weeks under illumination. D. Detail of green roots after removal of shoots just under the shoot/root junction.


      Figure 3. Detail of long-handled scissors used for detaching roots


      Figure 4. Detail of the coloration of detached root in response to dark (right) and light (left) exposure

Data analysis

The aim of this protocol is to increase the levels of carotenoids in roots. To evaluate the raise of these pigments two alternative methodologies could be used: on one hand, spectrophotometric analysis could be performed by many different protocols, evaluating in this case the total carotenoid content (i.e., Wellburn, 1994). Alternatively, detailed carotenoid composition could be achieved by liquid chromatography coupled to a diode array detector (HPLC-DAD) as detailed in Manzi et al., (2016). Carotenoid quantification should be performed accordingly to the method used.

Notes

Rates of growth may differ among citrus genotypes. To avoid unwanted effects on metabolism, avoid excessive growing of roots which may lead to an early senescence. To this, you might adjust the growing time of the plants accordingly (i.e., reducing the 3 week period under light conditions).
In order to obtain high levels of sample material a good option is increasing the number of individual roots rather than extending the root growing period. Consider that 20 roots provide approximately 1.5-2.0 g of fresh tissue.

Recipes

  1. Mix A
    Dilute a mix of myo-inositol (100 mg L-1), pyridoxine-HCl (1.0 mg L-1), thiamine-HCl (0.2 mg L-1), nicotinic acid (0.5 mg L-1) and glycine (0.2 mg L-1) in distilled water
    Aliquot in 1.5 ml Eppendorf tubes
    Aliquots must be stored at -20 °C
    Note: One of this Eppendorf (1.5 ml) is added for each 1.5 L of growing medium
  2. Sodium hypochlorite solution (50.0%, v/v)
    Mix equal parts of pure sodium hypochlorite (100%) and distilled MiliQ water
    Tween® 20 wetting agent at 0.1% (v/v) is added to the final solution
    This solution can be either stored or prepared in the moment
    Note: The volume of solution to prepare depends on the number of seeds to disinfect. To avoid any harmful effect of the hypochlorite, seeds must not be exposed to the disinfectant solution for more than 10 min
  3. NaOH (0.1 N)
    Dissolve 40 g of NaOH in 1.0 L of distilled water to make a 1.0 N solution
    This solution must be stored at 4 °C
    At the time of preparation of 0.1 N solution, take 1.0 ml of 1.0 N NaOH solution and complete to 10 ml by adding 9.0 ml of distilled water
    Note: Add drop by drop of 0.1 N NaOH to adjust the pH of the germination medium. Usually, only few drops are sufficient to achieve a pH = 5.7 ± 0.1 of 1.0 L of medium

Acknowledgments

This work was supported by the Ministerio de Economia (MINECO) and Universitat Jaume I through grants No. AGL2013- 42038-R and P1IB2013-23, respectively. MM was recipient of a ‘Santiago Grisolia’ fellowship from Generalitat Valenciana (Spain). This protocol is based on the methodology used in the manuscript Manzi et al. (2016).

References

  1. Arbona, V., Manzi, M., Ollas, C. and Gomez-Cadenas, A. (2013). Metabolomics as a tool to investigate abiotic stress tolerance in plants. Int J Mol Sci 14(3): 4885-4911.
  2. Cao, H., Wang, J., Dong, X., Han, Y., Ma, Q., Ding, Y., Zhao, F., Zhang, J., Chen, H., Xu, Q., Xu, J. and Deng, X. (2015). Carotenoid accumulation affects redox status, starch metabolism, and flavonoid/anthocyanin accumulation in citrus. BMC Plant Biol 15: 27.
  3. Lado, J., Rodrigo, M. J., López-Climent, M., Gómez-Cadenas, A. and Zacarías, L. (2016). Implication of the antioxidant system in chilling injury tolerance in the red peel of grapefruit. Postharvest Biol Tec 111: 214-223.
  4. Manzi, M., Lado, J., Rodrigo, M. J., Arbona, V. and Gomez-Cadenas, A. (2016). ABA accumulation in water-stressed Citrus roots does not rely on carotenoid content in this organ. Plant Sci 252: 151-161.
  5. Wellburn, A. R. (1994). The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol 144: 307–313.

简介

植物中的类胡萝卜素起着几个关键作用,例如作为轻收割机,抗氧化剂(Lado等人,2016),或作为角闪石内酯,脱落酸,挥发物和其他信号传导化合物的前体(Arbona et al。,2013)。虽然这些功能在光暴露的组织中是众所周知的,但是由于这些颜料的丰度降低,地下器官中的信息受到限制。为了更好地了解类胡萝卜素在根中的作用,我们开发了一种增加地下组织中这些颜料丰度的方法。我们利用了暴露于光的柑橘根发现色素沉淀以增加类胡萝卜素含量的事实。因此,这里我们描述一种增加柑橘根类胡萝卜素的简单方法。

背景 根中的类胡萝卜素丰度是非常有限的,因此,理解这些化合物的作用变得困难。根部对光线的曝光是增加这些组织中类胡萝卜素水平的简单,快速和有用的工具,特别是当与其他基因组方法相比较时,例如过度表达类胡萝卜素生物合成途径的一些关键基因(Cao等人,2015)。

关键字:脱落酸, 植物激素, 生长室, 体外培养, 根脱离, 种子萌发

材料和试剂

  1. Pirex ®培养管25 x 150 mm
  2. 一次性注射器(25 ml)
  3. 1.5 ml Eppendorf管
  4. 柑橘种子(例如,从商业砧木)
  5. Murashige和Skoog(MS)培养基(4,302.09mg L -1)(Duchefa Biochemie,目录号:M0221)
  6. 蔗糖(30g L -1 )(任何供应商)
  7. 无菌MiliQ水
  8. 琼脂(欧洲细菌琼脂)(Conda,Pronadisa,目录号:1800)
  9. (0.1%v/v)(Sigma-Aldrich,目录号:P2287)
  10. 肌醇(100mg L -1)(Duchefa Biochemie,目录号:I0609)
  11. 吡哆醇-HCl(1.0mg L -1)(Duchefa Biochemie,目录号:P0612)
  12. 硫胺素-HCl(0.2mg L -1)(Duchefa Biochemie,目录号:T0614)
  13. 烟酸(0.5mg L -1)(PANREACQUÍMICA,目录号:A0963)
  14. 甘氨酸(0.2mg L -1)(PANREACQUÍMICA,目录号:141340)
  15. 混合A(见配方)
  16. 次氯酸钠溶液(50.0%,v/v)(参见食谱)
  17. NaOH(0.1 N)(任何供应商)(见配方)

设备

  1. Pirex ®介质溶液瓶
  2. 高压灭菌器(Raypa,型号:蒸汽灭菌器AES-75)
  3. 层流罩(Bioquell,ASTEC MICROFLOW,型号:Microflow Laminar Flow Workstation M50546)
  4. 钩镊子
  5. 生长室(Snijders Scientific,型号:经济Lux气候室)
  6. 长柄剪刀带曲线尖端
  7. pH计(HACH LANGE西班牙,型号:pH计Basic 20)

程序

  1. 准备生长培养基
    1. 溶解4.3g L 的MS,1.0ml混合物A(参见食谱)和30g L -1的蔗糖在蒸馏水中。
    2. 用NaOH(0.1N)将溶液的pH调节至5.7±0.1
    3. 将溶液分配到瓶中进行高压灭菌。
    4. 将溶液在105°C下高压灭菌5分钟
    5. 溶解9.0 g L 琼脂琼脂固化培养基。
    6. 即使使用温热的介质和注射器,也可以将25ml溶液分配到培养管中并覆盖。
    7. 在115°C将含有管的机架高压灭菌15分钟。
    8. 让它冷却并在室温下储存。

  2. 植物材料制备
    1. 去除种子的外套以增加和均质种子发芽。避免损害种子;特别注意不要伤害胚胎(图1)。将其放入次氯酸钠溶液(50.0%,v/v)加0.1%(v/v)Tween 20分钟内灭菌10分钟。
    2. 用无菌蒸馏水冲洗种子三次,以除去次氯酸盐溶液
    3. 将种子存放在无菌层流罩中,防止脱水。


      图1.用于提高发芽率和均质化的种子包衣移除实例

  3. 萌发和生长条件
    1. 在无菌层流罩中并通过无菌镊子进行处理,将每根管子放入固化的生长培养基中并在播种后覆盖管(图2A)。
    2. 一旦将种子播种到生长室中,持续两周保持在25℃和黑暗以促进发芽(图2B)。
    3. 两周后,将发芽的幼苗(当时大约5厘米)转移到发光的生长室。将光周期条件设定为16:8小时(亮:暗),温度为25℃,最小辐射为150μmol/平方米以上光合作用辐射( PAR)(图2C)。
    4. 在光照条件下三周后植物生长并变成绿色,需要去除枝条。在无菌层流罩中,植物通过长柄剪刀脱离枝条(图3)。在层流罩的无菌条件下进行。通过在根/芽结合处下方约5毫米处切割出芽,将剩余的根保留在培养基中,以避免未来的芽再生(图2D)。
    5. 包含根组织的管在进行实验之前保持至少两天以排除伤口的任何副作用(图4)。


      图2.在体外条件下的幼苗发育实例 A.播种种子。 B.黑暗条件下生长的Etiolated幼苗。 C.照明3周后种子变绿。 D.去除芽/根结点下的枝条后,绿根的细节。


      图3.用于分离根的长柄剪刀的细节


      图4.响应暗(右)和轻(左)曝光分离根的着色细节

数据分析

该方案的目的是提高根中类胡萝卜素的含量。为了评估这些颜料的提高,可以使用两种替代方法:一方面,可以通过许多不同的方案进行分光光度分析,在这种情况下评估总胡萝卜素含量(,Wellburn,1994 )。或者,详细的类胡萝卜素组合物可以通过与二氧化硅阵列检测器(HPLC-DAD)相结合的液相色谱来实现,如Manzi等人(2016)所详述。应根据所使用的方法对类胡萝卜素进行定量。

笔记

柑橘基因型的增长率可能不同。为了避免不必要的对新陈代谢的影响,避免根部过度生长,这可能导致早期衰老。为此,您可以相应地调整植物生长时间(,即,在光线条件下减少3周的时间)。
为了获得高水平的样品材料,良好的选择是增加个体根的数量,而不是延长根系生长期。考虑到20个根提供约1.5-2.0g的新鲜组织。

食谱

  1. 混合A
    稀释肌醇(100mg/L),吡哆醇-HCl(1.0mg L -1),硫胺-HCl(0.2mg L -1) -1 ),烟酸(0.5mg L -1)和甘氨酸(0.2mg L -1)在蒸馏水中的溶液
    在1.5ml Eppendorf管中分批 等分试样必须储存于-20°C 注意:为每1.5升生长培养基添加其中一种Eppendorf(1.5毫升),
  2. 次氯酸钠溶液(50.0%,v/v)
    混合等份的纯次氯酸钠(100%)和蒸馏的MiliQ水 向最终溶液中加入20%0.1%(v/v)的润湿剂
    该解决方案可以存储或准备在
    的那一刻 注意:准备的溶液体积取决于要消毒的种子数量。为了避免次氯酸盐的任何有害影响,种子不能暴露于消毒剂溶液超过10分钟。
  3. NaOH(0.1N)
    将40g NaOH溶解在1.0L蒸馏水中,制成1.0N的溶液 此解决方案必须存储在4°C
    在制备0.1N溶液时,取1.0毫升1.0N NaOH溶液,加入9.0毫升蒸馏水,完成10ml。 注意:滴加0.1N NaOH滴加萌发培养基的pH值。通常,只有几滴足以达到1.0L培养基
    的pH = 5.7±0.1

致谢

这项工作由经济大臣(MINECO)和Jaume I大学分别通过拨款号AGL2013- 42038-R和P1IB2013-23支持。 MM由Generalitat Valenciana(西班牙)接受了"Santiago Grisolia"奖学金。该协议基于Manzi等人的手稿中使用的方法。 (2016)。

参考文献

  1. Arbona,V.,Manzi,M.,Ollas,C.and Gomez-Cadenas,A.(2013)。  代谢组学作为调查植物中非生物胁迫耐受的工具。 Int J Mol Sci 14(3):4885-4911 。
  2. Cao,H.,Wang,J.,Dong,X.,Han,Y.,Ma,Q.,Ding,Y.,Zhao,F.,Zhang,J.,Chen,H.,Xu, Xu,J. and Deng,X.(2015)。  类胡萝卜素积累影响柑橘中的氧化还原状态,淀粉代谢和类黄酮/花青素积累。 BMC Plant Biol 15:27.
  3. Lado,J.,Rodrigo,MJ,López-Climent,M.,Gómez-Cadenas,A.andZacarías,L。(2016)。  抗氧化系统对葡萄柚红皮中寒冷耐受性的影响 Postharvest Biol Tec 111 :214-223。
  4. Manzi,M.,Lado,J.,Rodrigo,MJ,Arbona,V.and Gomez-Cadenas,A.(2016)。  水分胁迫柑橘根中的ABA积累不依赖于该器官中的类胡萝卜素含量。植物科学 252: 151-161。
  5. Wellburn,AR(1994)。  叶绿素的光谱测定a和b,以及总类胡萝卜素,使用不同分辨率的分光光度计的各种溶剂。植物生理学144:307-313。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Manzi, M., Pitarch-Bielsa, M., Arbona, V. and Gómez-Cadenas, A. (2016). Protocol for Increasing Carotenoid Levels in the Roots of Citrus Plants. Bio-protocol 6(24): e2077. DOI: 10.21769/BioProtoc.2077.
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