Do isotopic respiratory signals trace changes in metabolic fluxes
Do isotopic respiratory signals trace changes in metabolic fluxes
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DOI：
10.1111/j.1469-8137.2010.03248.x
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发表时间：
2010-05
期刊：
影响因子：
9.4
通讯作者：
Christian Werner
中科院分区：
文献类型：
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作者：
Christian Werner
作为对Tcherkez（2010）的回应，我首先澄清了呼吸途径中的相对通量变化在多大程度上会导致呼吸CO2（dCres）同位素比值的显著变化;其次，为什么位置C-丙酮酸标记实验确实提供了这些通量变化的信息;第三，可以从这些类型的实验中获得生态相关的信息。Tcherkez（2010）质疑呼吸dCO 2的巨大变化源于碎片分馏，即丙酮酸脱羧（通过丙酮酸脱氢酶，PDH）后的代谢分支点，此时剩余的乙酰辅酶A可以进入克雷布斯循环（KC或TCA循环）或转化为次级代谢。首先，他的数字例子似乎与Rossmann等人（1991）报道的数据有些不一致，Rossmann等人（1991）在丙酮酸的C-2和C-3位置给出了21‰和27‰（而不是Tcherkez，2010）的25‰（总葡萄糖分子为25‰）。因此，0或100%承诺KC脱羧之间的理论最大同位素变化为4‰（C-1的21‰或全分子的25‰）。然而，在体内的KC脱羧的承诺可能会有所不同。KC是氨基酸生物合成的重要来源，为谷氨酸和天冬氨酸提供碳骨架，后者非常常见，蛋白质合成和周转需要大量的碳骨架（Hayes，2001）。如果丙酮酸没有完全呼吸，KC中会发生平衡和动力学同位素效应，导致呼吸的CO2和有机酸消耗（Tcherkez & Farquhar，2005）。正如Tcherkez（2010）所指出的，KC脱羧的低或高承诺都不会导致dCres的大幅变化。然而，在中间混合比，dCres可以降低到低于源dC签名。例如，当50%的乙酰辅酶A在KC中呼吸时，柠檬酸合酶的分馏（内在分馏）为23‰; Tcherkez & Farquhar，2005）导致有效同位素效应为11.6‰。进入KC的流量为50%的总反应则产生（）21‰ · 100 +（）27‰）11.6‰）· 50）27‰ · 50）<$200 =）26.9‰。此外，α-酮戊二酸脱氢酶也可能断裂（）23‰; Tcherkez & Farquhar，2005），因为反应由于转向氨基酸合成（特别是大多数富含C的氨基酸，Hayes，2001）而不完全。在上面的例子中，dCres将是）29.8‰。此外，如果反应不完全，PDH也可能断裂，这将进一步消耗丙酮酸的C-2位（例如，对于75%的PDH承诺，消耗4.5‰，Melzer &施密特，1987）。因此，通过PDH和KC的相对碳通量变化理论上可能会引起比Tcherkez（2010）所建议的更大的dCres变化（> 9‰）。然而，这些过程在体内知之甚少。然而，尽管如此，PDH或KC（即PDH/KC比）的相对脱羧速率变化可能无法完全解释dCres的观测动态，因为dCres的观测范围可能更大，克雷斯可能变得更正值（例如，冬青栎的dCres日变化为26.7至18.3‰，Halimium sp.为29.9至15.1‰）; Werner等人，2009; F. Wegener等人，未出版）。显著正的dCO 2比率可能来自变暗后苹果酸池的快速脱羧，其可能在光照期间积累，导致CO2的短光照后爆发（称为光增强暗呼吸（LEDR），Barthel等人，2007; Gessler等人，2009年）。苹果酸在C-4位富含C，其由磷酸烯醇丙酮酸羧化酶固定。然而，苹果酸酶肯定与同位素效应有关。如果我们假设动态瑞利过程（参见Gessler等人，2009），dCres将在变暗后立即被更多地耗尽，而随着苹果酸池的下降而变得更加富集（参见Werner等人，2009年）。这与在亮-暗转变期间观察到的dCres的动态相反（Werner等人，2007，2009; Barthel等人，2007年）。因此，到目前为止，这些机制不能完全解释dCres中的时间动态，并且可能叠加了多个过程（Werner等人，2009年）。也可能涉及其他途径，例如戊糖-磷酸途径（Mesellier et al.，2009年）。在动态代谢通量的分馏过程中仍然存在许多不确定性，仅用同位素质量平衡计算无法澄清这些问题。我们对碳同位素的同位素分馏和分子间分布的了解是基于对一两种植物（甚至酵母）的稀疏测量。因此，需要针对不同植物功能组中代谢通量和同位素特征的昼夜动态进行实验，以确定潜在的过程。Tcherkez（2010）进一步质疑位置标记实验的方法学方面：第一，标记吸收;第二，蒸腾速率的影响;第三，稀释效应。事实上，C-标记实验对实验条件如孵育时间、摄取和实验时机非常敏感。然而，如果在明确定义的条件下进行，位置C-标记实验提供非常可重复的结果。·在不同分子间位置标记的分子（即C-1或C-3丙酮酸盐）之间的吸收速率差异是非常不可能的，因为C溶液New Phytologist Letters Forum 569
In response to Tcherkez (2010) I clarify first, to what extent relative flux changes in the respiratory pathways can cause significant variations in the isotopic ratios of respired CO2 (dCres); second, why positional C-pyruvate labelling experiments do provide information on these flux changes; and third, that ecologically relevant information can be gained from these types of experiments. Tcherkez (2010) challenges that large changes in respired dCO2 originate from fragmentation fractionation, that is, from the metabolic branching point after the decarboxylation of pyruvate (by pyruvate dehydrogenase, PDH), when the remaining acetyl-CoA can either enter the Krebs cycle (KC or TCA cycle) or be converted into secondary metabolism. First, his numeric example seems somewhat inconsistent with the data reported by Rossmann et al. (1991), who give )21‰ at C-1 and )27‰ (not )25‰ as in Tcherkez, 2010) at C-2 and C-3 positions of pyruvate (the overall glucose molecule was )25‰). Therefore, the theoretical maximum isotopic variation between 0 or 100% commitment to KC decarboxylation is 4‰ (either )21‰ of C-1, or )25‰ of the full molecule). However, in vivo the commitment into KC decarboxylation may vary. The KC is an important source of amino acid biosynthesis, providing carbon skeletons for glutamic acid and aspartic acid, the latter being very common with large quantities required for protein synthesis and turnover (Hayes, 2001). If pyruvate is not fully respired, both equilibrium and kinetic isotope effects occur in the KC, leading to a depletion of both respired CO2 and the organic acids (Tcherkez & Farquhar, 2005). As pointed out by Tcherkez (2010), neither a low nor a high commitment into KC decarboxylation results in large changes in dCres. However, at intermediate mixing ratios, dCres can decrease below that of the source d C signature. For example, when 50% of the acetyl-CoA is respired in the KC, fractionation by the citrate synthase (intrinsic fractionation of )23‰; Tcherkez & Farquhar, 2005) results in an effective isotope effect of )11.6‰. The overall reaction with 50% flux into the KC then yields ()21‰ · 100 + ()27‰ )11.6‰) · 50 ) 27‰ · 50) ⁄ 200 = )26.9‰. Moreover, a-ketoglutarate dehydrogenase may also fractionate ()23‰; Tcherkez & Farquhar, 2005) because the reaction is incomplete as a result of the diversion to amino acid synthesis (notably the most C-enriched amino acids, Hayes, 2001). In the above example, dCres would be )29.8‰. Furthermore, PDH may also fractionate if the reaction is incomplete, which would further deplete the C-2 position of pyruvate (e.g. 4.5‰ depletion for a 75% commitment to the PDH, Melzer & Schmidt, 1987). Thus, relative carbon flux changes through PDH and the KC may theoretically induce much larger dCres variations (> 9‰) than suggested by Tcherkez (2010). However, little is known on these processes in vivo. Nevertheless, changes in the relative decarboxylation rates by PDH or the KC (i.e. the PDH ⁄ KC ratio) may not entirely explain the observed dynamics in dCres because the observed range in dCres can be larger and d Cres can become more positive than )21‰ (e.g. a diurnal variation of dCres of )26.7 to )18.3‰ in Quercus ilex and of )29.9 to )15.1‰ in Halimium sp.; Werner et al., 2009; F. Wegener et al., unpublished). Markedly positive dCO2 ratios may evolve from rapid decarboxylation of a malate pool after darkening, which may accumulate during the light, resulting in a short postillumination burst of CO2 (known as light enhanced dark respiration (LEDR), Barbour et al., 2007; Gessler et al., 2009). Malate is C-enriched in the C-4 position, which is fixed by phosphoenol pyruvate carboxylase. However, the malic enzyme is most certainly associated with an isotope effect. If we assume a dynamic Rayleigh process (see the equation in Gessler et al., 2009), dCres would be more depleted immediately after darkening while become more enriched as the malate pool declines (see Fig. 5 in Werner et al., 2009). This is inverse to the observed dynamics in dCres during light–dark transitions (Werner et al., 2007, 2009; Barbour et al., 2007). Hence, to date, these mechanisms cannot entirely explain the temporal dynamics in dCres and probably multiple processes are superimposed (Werner et al., 2009). Other pathways, such as the pentose-phosphate pathway, may also be involved (Bathellier et al., 2009). There are still many uncertainties in fractionation processes during dynamic metabolic fluxes, and isotopic mass-balance calculations alone will not clarify these issues. Our knowledge on the isotopic fractionation and intermolecular distribution of carbon isotopes is based on sparse measurements of one or two plant species (or even yeast). Hence, experiments addressing diurnal dynamics in metabolic fluxes and isotopic signatures in different plant functional groups are needed to identify the underlying processes. Tcherkez (2010) further questions the methodological aspects of positional labelling experiments: first, marker uptake; second, influence of transpiration rates; and third, dilution effects. Indeed, C-labelling experiments are very sensitive to experimental conditions such as incubation time, uptake and timing of the experiment. Nevertheless, if conducted under clearly defined conditions, positional C-labelling experiments deliver very reproducible results. • Differences in the absorption rates between molecules labelled at different intermolecular positions (i.e. C-1 or C-3 pyruvate) are very unlikely because the C solutions New Phytologist Letters Forum 569