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Extraction and Measurement the Activities of Cytosolic Phosphoenolpyruvate Carboxykinase (PEPCK) and Plastidic NADP-dependent Malic Enzyme (ME) on Tomato (Solanum lycopersicum)
番茄胞质中的磷酸烯醇丙酮酸羧激酶(PEPCK)和质体中的NADP依赖性苹果酸酶(ME)的提取和活性测定   

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Abstract

A recent study demonstrated that cytosolic phosphoenolpyruvate carboxykinase (PEPCK) and NADP-malic enzyme (NADP-ME) have an important role in malate metabolism during fruit ripening (Osorio et al., 2013). PEPCK catalyze the ATP-dependent decarboxylation of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) and NADP-ME, the reversible conversion of malate and pyruvate. Here, we present the detailed protocols to measure PEPCK activity in carboxylation direction by following oxidation of NADH and to measure NADP-ME activity based upon the reduction of NADP+.

Materials and Reagents

  1. PEPCK activity
    1. Un-harvested plant tissues (how to freeze the tissue is explained in the procedure section)
    2. Liquid N2
    3. Ice
    4. Bicine (Sigma-Aldrich, catalog number: B8660 )
    5. KOH
    6. EDTA (Sigma-Aldrich, catalog number: EDS-100G )
    7. Poly (ethylene glycol)-4000 (Sigma-Aldrich, catalog number: 81240 )
    8. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815 )
    9. β-nicotinamide adenine dinucleotide reduced form (NADH) (Roche Diagnostics, catalog number: 10128015001 )
    10. 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) (Sigma-Aldrich, catalog number: H3375 )
    11. KCl
    12. MnCl2
    13. Phosphoelnolpyryvate (PEP) (Bio Vectra, catalog number: 2552)
    14. Adenosine 5′-diphosphate sodium salt (ADP) (Sigma-Aldrich, catalog number: A2754 )
    15. KHCO3
    16. L-Malate dehydrogenase (Roche Diagnostics, catalog number: 10127248001 )
    17. Bradford stock solution (Bio-Rad Laboratories, catalog number, 500-0006 )
    18. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2058 )
    19. Extraction buffer 1 (see Recipes)
    20. Extraction buffer 2 (see Recipes)
    21. Buffer 3 (see Recipes)
    22. PEPCK assay mix 1 (see Recipes)

  2. NADP-ME activity
    1. Un-harvested plant tissues (how to freeze the tissue is explained in the procedure section)
    2. Liquid N2
    3. Ice
    4. Tris-Base (United State Biological, catalog number: T8600 )
    5. MnCl2
    6. EDTA (Sigma-Aldrich, catalog number: EDS-100G)
    7. Glycerol (Sigma-Aldrich, catalog number: G5516 )
    8. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
    9. β-nicotinamide adenine dinucleotide phosphate (NADP+) (Roche Diagnostics, catalog number: 10128058001 )
    10. L-Malate (Sigma-Aldrich, catalog number: M1000 )
    11. Bradford stock solution (Bio-Rad Laboratories, catalog number, 500-0006)
    12. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2058)
    13. Extraction buffer 4 (see Recipes)
    14. NADP-ME assay mix 1 (see Recipes)

Equipment

  1. Small mortar and pestle
  2. 2 ml and 1.5 ml microfuge tubes
  3. Pipettes
  4. Balance
  5. 2 ml centrifuge (Hettich Mikro 22R)
  6. 96 well polystyrene microplate (flat bottom) (Corning, catalog number: 3300 )
  7. A computer supported microplate spectrophotometer for kinetic (time-course) measurement mode (Elisa microplate-spetrophotometer) (BioTek Instruments, model: EL808 )

Procedure

  1. Extraction for measuring PEPCK activity
    This protocol applies to extraction of PEPCKs from plant tissues.
    1. Collect the sample and freeze immediately in liquid N2. Store at -80 °C until use.
    2. Add small amount of liquid N2 and frozen sample into a mortar. Grind the tissue until the sample is a fine powder. Carefully add more liquid N2 if needed to keep frozen.
    3. Weight 500 mg of tissue into microfuge tube and add 900 µl of extraction buffer 1 (EB1) (for fruit tissue the ratio of powered tissue/EB is 1:1.8, w/v. This ratio may need to be increased to 1:3 for leaf tissue).
    4. Keep on ice while preparing all samples and vortex for 30 sec.
    5. Centrifuge 15 min at 4 °C and 13,000 x g. Remove 500 µl supernatant (clarified extract) to a fresh microfuge tube and add 850 µl of extraction buffer 2 (EB2), to give a final concentration of 35% PEG (mix well).
    6. Incubate on ice for 10 min and centrifuge for 20 min at 13,000 x g. The supernatant is discarded.
    7. Re-suspend the pellet in 100 µl of buffer 3 (B3) and keep on ice. Measure immediately PEPCK activity or the aliquots of clarified extracts can be snap frozen in liquid N2 and stored at -80 °C for future use.

  2. PEPCK activity
    The activity of PEPCK is measured in carboxylation direction by following oxidation of NADH at 340 nm at 25 °C. Briefly, a continuous assay is used in which OAA produced by PEPCK is immediately reduced to malate, and this is achieved by the inclusion of malate dehydrogenase. The oxidation of NADH by malate dehydrogenase is measured at 340 nm using a spectrophotometer.
    1. Accurately pipette 2-10 µl of clarified extract into a microplate well.
    2. Use repeat pipetor to add 150 µl PEPCK assay mix 1 to each well and immediately place in microplate spectrophotometer. Continuously monitor NADH oxidation to NAD+ as a decline in absorbance at 340 nm (A340), taking readings every 5-10 sec for up to 5 min (the samples need to shake while reading).
    3. Correct for background NADH oxidation by omitting PEP from the reaction mixture. Ensure that the decline in A340 (amount of NADH being oxidized; ε340 = 6,220/M/cm) is proportional to assay time and concentration of enzyme assayed. Dilution of clarified extract in extraction buffer may be necessary for samples containing abundant PEPCK activity.
      Note: One international unit (U) of enzyme activity is defined as the amount of enzyme resulting in the production of 1 µmol of product per min at 25 °C. PEPCK activity in (U/ml clarified extract) = (ΔA340/min x clarified extract dilution factor)/6.22. Thus, if 3.0 μl of clarified extract mixed with 150 μl of PEPCK reaction mixture yields a ΔA340/min of 0.2 at 340 nm, then the PEPCK activity = (0.2 x 50)/6.22 U/ml = 1.6 U/ml.

  3. Extraction for measuring NADP-ME activity
    This protocol applies to extraction of NADP-MEs from plant tissues.
    1. Collect the sample and freeze immediately in liquid N2. Store at -80 °C until use.
    2. Add small amount of liquid N2 and frozen sample into a mortar. Grind the tissue until the sample is a fine powder. Carefully add more liquid N2 if needed to keep frozen.
    3. Weight 100 mg of tissue into microfuge tube and add 200 µl of extraction buffer 4 (EB4) (for fruit tissue the ratio of powered tissue/EB is 1:2, w/v. This ratio may need to be increased to 1:3 for leaf tissue).
    4. Keep on ice while preparing all samples and vortex for 30 sec.
    5. Centrifuge 10 min at 4 °C and 13,000 x g. Transfer the supernatant to a fresh microfuge tube. Measure NADP-ME activity immediately or the aliquots of clarified extracts can be snap frozen in liquid N2 and stored at -80 °C for future use.

  4. NADP-ME activity
    The activity of NADP-ME is based upon the reduction of NADP+ at 340 nm at 25 °C. Briefly, a continuous assay is used in which malate is oxidized to pyruvate and CO2, and NADP+ is reduced to NADPH. The reduction of NADP+ by NADP-ME is measured at 340 nm using a spectrophotometer.
    1. Accurately pipette 5-10 µl of clarified extract into a microplate well.
    2. Use repeat pipetor to add 100 µl NADP-ME assay mix 1 to each well and immediately place in microplate spectrophotometer. Continuously monitor NADP+ reduction to NADPH as an increase in absorbance at 340 nm (A340), taking readings every 5-10 sec for up to 5 min (the samples need to shake while reading).
    3. Correct for background NADP+ reduction by omitting L-malate from the reaction mixture. Ensure that the increase in A340 is proportional to assay time and concentration of enzyme assayed. Dilution of clarified extract in extraction buffer may be necessary for samples containing abundant NADP-ME activity.
      Note: One international unit (U) of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 µmol of NADPH/min under the specified conditions. NADP-ME activity in (U/ml clarified extract) = (ΔA340/min x clarified extract dilution factor)/6.22. Thus, if 2.0 μl of clarified extract mixed with 100 μl of NADP-ME reaction mixture yields a ΔA340/min of 1.5 at 340 nm, then the NADP-ME activity = (1.5 x 50)/6.22 U/ml = 12.5 U/ml.

  5. Bradford assay of protein concentration
    1. For preparing the BSA standard curve:
      Make 1 ml stock solution of 10 mg BSA/200 ml phosphate buffered saline (PBS) (pH 7.4) and prepare a duplicate standard curve, using template below:

      Final concentration (ng/µl)   
      Vol. (µl) BSA stock (0.05 mg/ml)
      PBS (µl)
      µg protein/well
      0
      0
      200
      0
      6.25
      25
      175
      1.25
      12.5
      50
      150
      2.5
      18.7
      75
      125
      3.75
      25.0
      100
      100
      5

    2. Pipette 50 µl of aliquot supernatants (or its dilutions) or BSA standards to each well.
    3. Add 130 µl Bradford stock solution (see Materials and Reagents section) previously dissolved in distillate water (dilution 1/5).
    4. Shake briefly and wait 5 min. Determinate the OD at 595 nm. Use slope and blank obtained from the calibration to determinate the protein concentration in each extracts.

Recipes

  1. Extraction buffer 1 (EB1, kept on ice)
    500 mM Bicine-KOH (pH 9.0)
    200 mM KCl
    3 mM EDTA
    5% (w/v) PEG-4000
    25 mM DTT (add freshly every time before using it)
    0.4% BSA
  2. Extraction buffer 2 (EB 2, kept on ice)
    500 mM Bicine-KOH (pH 9.0)
    3 mM EDTA
    55% (w/v) PEG-4000
    25 mM DTT (add freshly every time before using it)
  3. Buffer 3 (EB3, kept on ice)
    10 mM Bicine-KOH (pH 9.0)
    25 mM DTT (add freshly every time before using it)
    Note: EB1, EB2, and EB3 without DTT can be kept at 4 ºC for few weeks.
  4. PEPCK assay mix 1
    100 mM Hepes-KOH (pH 6.8)
    100 mM KCl
    0.14 mM NADH
    25 mM DTT (add freshly every time before using it)
    6 mM MnCl2
    6 mM PEP
    1 mM ADP
    90 mM KHCO3
    6 U ml-1 malate dehydrogenase
  5. Extraction buffer 4 (EB4, kept on ice)
    100 mM Tris-HCl (pH 8.0)
    5 mM MgCl2
    2 mM EDTA
    10% (v/v) glycerol
    10 mM 2-mercaptoethanol
  6. NADP-ME assay mix 1 (kept on ice)
    50 mM Tris-HCl (pH 8.0)
    10 mM MgCl2
    0.5 mM NADP+
    10 mM L-malate

Acknowledgments

We acknowledge the excellent care of the plants by Helga Kulka (Max- Planck-Institut für Molekulare Planzenphysiologie) and Hanna Levanony (Weizmann Institute of Science). This protocol was adapted from Osorio et al. (2013).

References

  1. Centeno, D. C., Osorio, S., Nunes-Nesi, A., Bertolo, A. L., Carneiro, R. T., Araujo, W. L., Steinhauser, M. C., Michalska, J., Rohrmann, J., Geigenberger, P., Oliver, S. N., Stitt, M., Carrari, F., Rose, J. K. and Fernie, A. R. (2011). Malate plays a crucial role in starch metabolism, ripening, and soluble solid content of tomato fruit and affects postharvest softening. Plant Cell 23(1): 162-184.
  2. Osorio, S., Vallarino, J. G., Szecowka, M., Ufaz, S., Tzin, V., Angelovici, R., Galili, G. and Fernie, A. R. (2013). Alteration of the interconversion of pyruvate and malate in the plastid or cytosol of ripening tomato fruit invokes diverse consequences on sugar but similar effects on cellular organic acid, metabolism, and transitory starch accumulation. Plant Physiol 161(2): 628-643.

简介

最近的研究表明胞质磷酸烯醇丙酮酸羧激酶(PEPCK)和NADP-苹果酸酶(NADP-ME)在果实成熟期间在苹果酸代谢中具有重要作用(Osorio等人,2013)。 PEPCK催化草酰乙酸(OAA)向磷酸烯醇丙酮酸(PEP)和NADP-ME的ATP依赖性脱羧,苹果酸和丙酮酸的可逆转化。 在这里,我们提出详细的协议,以测量PEPCK活性的羧化方向通过以下氧化NADH和基于NADP + 的还原测量NADP-ME活性。

材料和试剂

  1. PEPCK活性
    1. 未收割的植物组织(如何冻结组织在程序部分解释)
    2. 液体N <2>
    3. 冰块
    4. Bicine(Sigma-Aldrich,目录号:B8660)
    5. KOH
    6. EDTA(Sigma-Aldrich,目录号:EDS-100G)
    7. 聚(乙二醇)-4000(Sigma-Aldrich,目录号:81240)
    8. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:43815)
    9. β-烟酰胺腺嘌呤二核苷酸还原型(NADH)(Roche Diagnostics,目录号:10128015001)
    10. 4-(2-羟乙基)哌嗪-1-乙磺酸,N-(2-羟乙基)哌嗪-N' - (2-乙磺酸)(HEPES)(Sigma-Aldrich,目录号:H3375)
    11. KCl
    12. MnCl 2
    13. 磷酸芘酯(PEP)(Bio Vectra,目录号:2552)
    14. 腺苷5'-二磷酸钠盐(ADP)(Sigma-Aldrich,目录号:A2754)
    15. KHCO 3
    16. L-苹果酸脱氢酶(Roche Diagnostics,目录号:10127248001)
    17. Bradford储液(Bio-Rad Laboratories,目录号,500-0006)
    18. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2058)
    19. 提取缓冲液1(参见配方)
    20. 提取缓冲液2(参见配方)
    21. 缓冲区3(参见配方)
    22. PEPCK测定混合1(参见配方)

  2. NADP-ME活性
    1. 未收割的植物组织(如何冻结组织在程序部分解释)
    2. 液体N <2>
    3. 冰块
    4. Tris-Base(United States Biological,目录号:T8600)
    5. MnCl 2
    6. EDTA(Sigma-Aldrich,目录号:EDS-100G)
    7. 甘油(Sigma-Aldrich,目录号:G5516)
    8. 2-巯基乙醇(Sigma-Aldrich,目录号:M6250)
    9. β-烟酰胺腺嘌呤二核苷酸磷酸(NADP + )(Roche Diagnostics,目录号:10128058001)
    10. L-苹果酸盐(Sigma-Aldrich,目录号:M1000)
    11. Bradford储液(Bio-Rad Laboratories,目录号,500-0006)
    12. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2058)
    13. 提取缓冲液4(参见配方)
    14. NADP-ME测定混合物1(参见配方)

设备

  1. 小砂浆和杵
  2. 2 ml和1.5 ml微量离心管
  3. 移液器
  4. 余额
  5. 2ml离心机(Hettich Mikro 22R)
  6. 96孔聚苯乙烯微板(平底)(Corning,目录号:3300)
  7. 计算机支持的用于动力学(时间过程)测量模式的微孔板分光光度计(Elisa微孔板 - 分光光度计)(BioTek Instruments,型号:EL808)

程序

  1. 提取测量PEPCK活性
    该协议适用于从植物组织中提取PEPCK
    1. 收集样品并立即在液体N 2中冷冻。 储存于-80℃直至使用。
    2. 将少量液体N 2和冷冻样品加入研钵中。 研磨组织直到样品是细粉末。 小心地加入更多的液体N <2>如果需要保持冷冻
    3. 将500mg组织重量500mg装入微量离心管中并加入900μl提取缓冲液1(EB1)(对于果实组织,动力组织/EB的比例为1:1.8,w/v),该比例可能需要增加至1:3 对于叶组织)
    4. 在准备所有样品的同时保持在冰上,并涡旋30秒
    5. 在4℃和13,000×g离心15分钟。删除500微升上清液(澄清的提取物)到一个新鲜的微量离心管,加入850微升提取缓冲液2(EB2),使最终浓度为35%的PEG(混合良好)。
    6. 在冰上孵育10分钟,并在13,000×g离心20分钟。弃去上清液。
    7. 重悬浮在100微升缓冲液3(B3),并保持在冰上的沉淀。立即测量PEPCK活性或可将澄清提取物的等分试样快速冷冻在液体N 2中并储存在-80℃以备将来使用。

  2. PEPCK活动
    PEPCK的活性通过在25℃下在340nm下NADH的氧化后在羧化方向上测量。简言之,使用连续测定法,其中由PEPCK产生的OAA立即还原成苹果酸盐,并且这通过包含苹果酸脱氢酶来实现。使用分光光度计在340nm下测量苹果酸脱氢酶对NADH的氧化
    1. 准确吸取2-10μl澄清的提取物到微孔板孔中
    2. 使用重复移液器添加150μlPEPCK测定混合1每个孔,立即放置在微孔板分光光度计。连续监测NADH氧化为NAD + ,作为在340nm(A 340)的吸光度的下降,每5-10秒读取最多5分钟(样品需要在阅读时震动)。
    3. 通过从反应混合物中省略PEP来校正背景NADH氧化。确保A 340(NADH被氧化的量;ε340 = 6,220/M/cm)的下降与测定时间和测定的酶浓度成比例。对于含有丰富的PEPCK活性的样品,澄清提取物在提取缓冲液中的稀释可能是必需的。
      注意:酶活性的一个国际单位(U)定义为在25℃下每分钟产生1μmol产物的酶量。 PEPCK活性(U/ml澄清提取物)=(ΔA/340×min /澄清提取物稀释因子)/6.22。因此,如果3.0μl澄清的提取物与150μlPEPCK反应混合物混合,则在340nm下产生0.2的ΔAinf 340 340,这样,在340nm处的ΔA<340> PEPCK活性=(0.2×50)/6.22U/ml = 1.6U/ml。

  3. 测量NADP-ME活性的提取
    该协议适用于从植物组织中提取NADP-ME
    1. 收集样品并立即在液体N 2中冷冻。储存于-80℃直至使用。
    2. 将少量液体N 2和冷冻样品加入研钵中。研磨组织直到样品是细粉末。小心地加入更多的液体N <2>如果需要保持冷冻
    3. 重量将100mg组织放入微量离心管中并加入200μl提取缓冲液4(EB4)(对于果实组织,动力组织/EB的比例为1:2,w/v),该比例可能需要增加至1:3对于叶组织)
    4. 在准备所有样品的同时保持在冰上,并涡旋30秒
    5. 在4℃和13,000×g离心10分钟。转移上清液到一个新鲜的微量离心管。立即测量NADP-ME活性或者可以将澄清的提取物的等分试样快速冷冻在液体N 2中并储存在-80℃以备将来使用。

  4. NADP-ME活动
    NADP-ME的活性基于在340nm下在25℃下NADP +的还原。简言之,使用连续测定法,其中将苹果酸盐氧化成丙酮酸盐和CO 2 2,并将NADP +还原成NADPH。使用分光光度计在340nm测量NADP-ME对NADP + 的还原。
    1. 准确吸取5-10微升澄清的提取物到微孔板孔
    2. 使用重复移液器添加100微升NADP-ME测定混合1到每个孔,并立即放置在微孔板分光光度计。作为在340nm处的吸光度的增加(A 340),连续监测NADP +还原为NADPH,每5-10秒读取读数直到5分钟(样品需要在阅读时震动)。
    3. 通过从反应混合物中省略L-苹果酸盐来校正背景NADP + 还原。确保A 340的增加与测定的酶的测定时间和浓度成比例。对于含有丰富的NADP-ME活性的样品,澄清提取物在提取缓冲液中的稀释可能是必需的。
      注意:酶活性的一个国际单位(U)定义为催化形成1μmolNADPH/min 的酶的量。 在指定的条件下。 NADP-ME活性(U/ml 澄清的提取物)=(ΔA /min x澄清的提取物稀释因子)/6.22。 因此,如果2.0μl澄清的提取物与100μlNADP-ME反应混合物混合,在340nm处产生1.5的ΔA<340> /min ,则NADP-ME活性=(1.5×50)/6.22U/ml = 12.5U/ml。

  5. Bradford蛋白浓度测定
    1. 对于制备BSA标准曲线:
      制备1ml 10mg BSA/200ml磷酸盐缓冲盐水(PBS)(pH 7.4)的储备溶液,并使用以下模板制备重复的标准曲线:

      最终浓度(ng /μl)   
      Vol。 (μl)BSA原液(0.05mg/ml)
      PBS(μl)
      μg蛋白/孔
      0
      0
      200
      0
      6.25
      25
      175
      1.25
      12.5
      50
      150
      2.5
      18.7
      75
      125
      3.75
      25.0
      100
      100
      5

    2. 每孔加入50μl等分试样上清液(或其稀释液)或BSA标准品
    3. 加入130μl布氏原液(见材料和试剂部分),先前溶于蒸馏水(稀释1/5)。
    4. 短暂摇动,等待5分钟。 测定595nm处的OD。 使用从校准中获得的斜率和空白来确定每种提取物中的蛋白质浓度

食谱

  1. 提取缓冲液1(EB1,保存在冰上)
    500mM N-二甘氨酸-KOH(pH9.0) 200 mM KCl
    3 mM EDTA
    5%(w/v)PEG-4000
    25 mM DTT(每次使用前都要新鲜加入)
    0.4%BSA
  2. 提取缓冲液2(EB 2,保存在冰上)
    500mM N-二甘氨酸-KOH(pH9.0) 3 mM EDTA
    55%(w/v)PEG-4000
    25 mM DTT(每次使用前都要新鲜加入)
  3. 缓冲液3(EB3,保存在冰上)
    10mM Bicine-KOH(pH9.0) 25 mM DTT(每次使用前都要新鲜加入)
    注意:没有DTT的EB1,EB2和EB3可以在4ºC保存几周。
  4. PEPCK测定混合1
    100mM Hepes-KOH(pH 6.8)
    100 mM KCl
    0.14mM NADH
    25 mM DTT(每次使用前都要新鲜加入)
    6mM MnCl 2
    6 mM PEP
    1 mM ADP
    90mM KHCO 3
    6U ml 苹果酸脱氢酶
  5. 提取缓冲液4(EB4,保存在冰上)
    100mM Tris-HCl(pH8.0) 5mM MgCl 2/
    2mM EDTA 10%(v/v)甘油 10mM 2-巯基乙醇
  6. NADP-ME测定混合物1(保存在冰上)
    50mM Tris-HCl(pH8.0)
    10mM MgCl 2/
    0.5 mM NADP +
    10mM L-苹果酸盐

致谢

我们承认Helga Kulka(Max-Planck-InstitutfürMolekulare Planzenphysiologie)和Hanna Levanony(Weizmann Institute of Science)对植物的优良护理。该协议改编自Osorio等人(2013)。

参考文献

  1. Centeno,DC,Osorio,S.,Nunes-Nesi,A.,Bertolo,AL,Carneiro,RT,Araujo,WL,Steinhauser,MC,Michalska,J.,Rohrmann,J.,Geigenberger, ,Stitt,M.,Carrari,F.,Rose,JKand Fernie,AR(2011)。 苹果酸在番茄果实的淀粉代谢,成熟和可溶性固体含量中起着至关重要的作用,并影响采后植物细胞 23(1):162-184。
  2. Osorio,S.,Vallarino,J.G。,Szecowka,M.,Ufaz,S.,Tzin,V.,Angelovici,R.,Galili,G.and Fernie,A.R。 在成熟番茄果实的质体或细胞溶质中改变丙酮酸和苹果酸的相互作用引起了不同的后果糖但对细胞有机酸,代谢和瞬时淀粉积累的相似作用。植物生理学161(2):628-643。
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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. Osorio, S., Vallarino, J. G., Szecowka, M., Ufaz, S., Tzin, V., Angelovici, R., Galili, G. and Fernie, A. R. (2014). Extraction and Measurement the Activities of Cytosolic Phosphoenolpyruvate Carboxykinase (PEPCK) and Plastidic NADP-dependent Malic Enzyme (ME) on Tomato (Solanum lycopersicum). Bio-protocol 4(9): e1122. DOI: 10.21769/BioProtoc.1122.
  2. Osorio, S., Vallarino, J. G., Szecowka, M., Ufaz, S., Tzin, V., Angelovici, R., Galili, G. and Fernie, A. R. (2013). Alteration of the interconversion of pyruvate and malate in the plastid or cytosol of ripening tomato fruit invokes diverse consequences on sugar but similar effects on cellular organic acid, metabolism, and transitory starch accumulation. Plant Physiol 161(2): 628-643.
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