Review 回顾
Interdependence between chloroplasts and mitochondria in the light and the dark
叶绿体和线粒体在光明和黑暗中的相互依存关系

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Keywords 关键字

Chloroplast
Chlororespiration
Excess reductant
Metabolite exchange
Mitochondrion
Photosynthesis
Respiration

叶绿体
氯呼吸
过量还原剂
代谢物交换
线粒体
光合作用
呼吸

Abbreviations 缩写

CR, chlororespiration
DHAP, dihydroxyacetone phosphate
ETC, electron transport chain
Fd, ferredoxin
G6PDH, glucose-6-P dehydrogenase
GAPDH, glyceraldehyde-3-P dehydrogenase
LEDR, light enhanced dark respiration
LHC, light harvesting complex
Mal, malate
MDH, malate dehydrogenase
ME, malic enzyme
NR, nitrate reductase
OAA, oxaloacetate
2-OG, 2-oxoglutarate
PDC, pyruvate dehydrogenase complex
PEP, phosphoenol pyruvate
PEPC, PEP carboxylase
3-PGA, 3-phosphoglycerate
PGK, phosphoglycerate kinase
PIB, post-illumination burst
PK, pyruvate kinase
PQ, plastoquinone
PSI, photosystem I
PSII, photosystem II
RuBP, ribulose 1,5-bisphosphate
SHAM, salicylhydroxamic acid
TCA, tricarboxylic acid
Td, thioredoxin
TP, triose phosphate

CR、氯呼吸
DHAP、磷酸二羟基丙酮
ETC、电子传递链
Fd、铁氧还蛋白
G6PDH、葡萄糖-6-P 脱氢酶
GAPDH、甘油醛-3-P 脱氢酶
LEDR、光增强暗呼吸
LHC、光捕获复合物
Mal、苹果酸
MDH、苹果酸脱氢酶
ME、苹果酸酶
NR、硝酸还原酶
OAA、草酰乙酸
2-OG、2-氧代戊二酸
PDC、丙酮酸脱氢酶复合物
PEP、磷酸烯醇丙酮酸
PEPC、PEP 羧化酶
3-PGA、3-磷酸甘油酸
PGK、磷酸甘油酸激酶
PIB、照明后突发
PK、丙酮酸激酶
PQ、质体醌
PSI、光系统 I
PSII、光系统 II
RuBP、核酮糖 1,5-二磷酸
SHAM、水杨异羟肟酸
TCA、三羧酸
Td、硫氧还蛋白
TP、磷酸丙糖

1. Introduction 1. 引言

Plants grow using light energy to photosynthetically convert atmospheric CO2 into carbon-rich compounds (e.g. carbohydrates) in the chloroplasts. These compounds are then respired in the cytosol and mitochondria to generate the energy and carbon intermediates necessary for biosynthesis. The two processes are interdependent, with respiration relying on photosynthesis for substrate whereas cellular photosynthesis depends on respiration for a range of compounds (e.g. ATP; see later sections). Surprisingly, however, most researchers study the two processes independently. In this review, we discuss the interdependence of chloroplasts and mitochondria. The mechanisms by which common metabolites are exchanged between chloroplasts and mitochondria via the cytosol are first discussed. The review then assesses the role of mitochondria in the light. Finally, it discusses the interaction between mitochondria and chloroplasts in darkness and the phenomenon of chlororespiration.
植物利用光能生长,将大气中的 CO2 光合作用转化为叶绿体中富含碳的化合物(例如碳水化合物)。然后,这些化合物在胞质溶胶和线粒体中呼吸,以产生生物合成所需的能量和碳中间体。这两个过程是相互依赖的,呼吸作用依赖于光合作用作为底物,而细胞光合作用依赖于一系列化合物(例如 ATP;见后面的章节)的呼吸作用。然而,令人惊讶的是,大多数研究人员独立研究这两个过程。在这篇综述中,我们讨论了叶绿体和线粒体的相互依赖性。首先讨论了通过胞质溶胶在叶绿体和线粒体之间交换常见代谢物的机制。然后,本综述评估了线粒体在光线下的作用。最后,它讨论了黑暗中线粒体和叶绿体之间的相互作用以及氯呼吸现象。

2. Interactions between organelles depends on metabolite exchange
2. 细胞器之间的相互作用取决于代谢物交换

Interactions between chloroplasts and mitochondria depend on exchange of metabolites such as ATP (energy), NAD(P)H (reducing equivalents) and carbon skeletons. Some metabolites are transported across membranes of the organelles by specific translocators, whereas others are transported by metabolite shuttles because they cannot be translocated directly. Metabolite shuttles may also serve multiple functions such as transferring both ATP and/or reducing equivalents or carbon skeletons. In this section we outline the ways in which metabolites are transported across organelle membranes.
叶绿体和线粒体之间的相互作用取决于代谢物的交换,例如 ATP(能量)、NAD(P)H(还原当量)和碳骨架。一些代谢物通过特定的转运物跨细胞器膜运输,而另一些代谢物则通过代谢物穿梭运输,因为它们不能直接易位。代谢物穿梭物还可以具有多种功能,例如转移 ATP 和/或还原当量或碳骨架。在本节中,我们概述了代谢物跨细胞器膜运输的方式。

2.1. ATP exchange 2.1. ATP 交易所

The highly active mitochondrial ATP/ADP translocator rapidly exports ATP from the matrix to the cytosol in exchange for ADP [1] (Fig. 1). In contrast, the activity and affinity of the chloroplast translocator are very low [2, 3] and possibly only active in young chloroplasts to import ATP [4].
高度活跃的线粒体 ATP/ADP 转运蛋白将 ATP 从基质快速输出到胞质溶胶中,以换取 ADP [1](图 1)。相比之下,叶绿体转运体的活性和亲和力非常低 [23],并且可能仅在年轻的叶绿体中具有输入ATP的活性[4]。
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Fig. 1. ATP exchanges among chloroplasts, the cytosol and mitochondria. ETC, electron transport chain; GP, NADP+-GAPDH (glyceraldehyde-3-P dehydrogenase); GPK, PGK (phosphoglycerate kinase) and NAD+GAPDH (glyceraldehyde-3-P dehydrogenase); PEPC, PEP carboxylase; PK, pyruvate kinase.
图 1.叶绿体、胞质溶胶和线粒体之间的 ATP 交换。ETC, 电子传递链;GP、NADP+-GAPDH(甘油醛-3-P 脱氢酶);GPK、PGK(磷酸甘油酸激酶)和 NAD+GAPDH(甘油醛-3-P 脱氢酶);PEPC,PEP 羧化酶;PK,丙酮酸激酶。

Chloroplast ATP exchange can also occur via the dihydroxyacetone 3-phosphate (DHAP)/3-phosphoglycerate (3-PGA) shuttle, using the phosphate translocator of the chloroplast membrane [5] (Fig. 1). The conversion of 3-PGA to DHAP in the chloroplast consumes ATP and NADPH, which are regenerated in the cytosol by the NAD+-dependent, phosphorylating GAPDH/PGA-kinase (Fig. 1). However, no ATP is exported when cytosolic DHAP is converted to 3-PGA by the NADP+-dependent non-phosphorylating GAPDH/PGK, which produces NADPH only (Fig. 1).
叶绿体 ATP 交换也可以通过使用叶绿体膜的磷酸盐转运点 [5] 通过三磷酸二羟基丙酮 (DHAP)/3-磷酸甘油酸酯 (3-PGA) 穿梭进行(图 1)。叶绿体中 3-PGA 转化为 DHAP 会消耗 ATP 和 NADPH,它们在胞质溶胶中通过 NAD+ 依赖性磷酸化 GAPDH/PGA 激酶再生(图 1)。然而,当胞质 DHAP 被 NADP+ 依赖性非磷酸化 GAPDH/PGK 转化为 3-PGA 时,不会输出 ATP,而 NADPH 仅产生 NADPH(图 1)。
Although these shuttles are capable of transporting both NADPH and ATP, they do not appear to export significant quantities of ATP under physiological conditions, as the non-phosphorylating system predominates [6]. The DHAP/3-PGA shuttle therefore utilises chloroplastic ATP and exports reducing equivalents from the chloroplast [6].
尽管这些穿梭车能够运输 NADPH 和 ATP,但在生理条件下,它们似乎不会输出大量的 ATP,因为非磷酸化系统占主导地位 [6]。因此,DHAP/3-PGA 穿梭物利用叶绿体 ATP 并从叶绿体中输出还原当量 [6]。
Import of ATP by this shuttle is probably more efficient, because DHAP can be converted to PGA via only one route (Fig. 1) which yields both NADPH and ATP. In chloroplasts, isolated from a mutant of Chlamydomonas deficient in the chloroplast ATP synthase, the DHAP/3-PGA shuttle had a much larger capacity for ATP import than the ATP translocator [7]. In these illuminated chloroplasts protein synthesis was highly stimulated by DHAP and GAP (5-fold) but less so by ATP (2-fold). On the other hand, 3-PGA strongly inhibited protein synthesis. Protein synthesis in the wild-type chloroplasts was not affected by these metabolites.
通过这种穿梭车导入 ATP 可能更有效,因为 DHAP 只能通过一条途径转化为 PGA(图 1),从而产生 NADPH 和 ATP。在叶绿体中,从缺乏叶绿体 ATP 合酶的衣藻突变体中分离出的叶绿体中,DHAP/3-PGA 穿梭机的 ATP 输入能力比 ATP 转运蛋白大得多 [7]。在这些照射的叶绿体中,DHAP 和 GAP (5 倍) 对蛋白质合成的刺激很大,但 ATP (2 倍) 的刺激较小。另一方面,3-PGA 强烈抑制蛋白质合成。野生型叶绿体中的蛋白质合成不受这些代谢物的影响。
In summary, chloroplasts exhibit a far lower capacity for ATP export than mitochondria.
总之,叶绿体的 ATP 输出能力远低于线粒体。

2.2. Transport of reducing equivalents across membranes
2.2. 还原当量跨膜的传输

NAD(P)H cannot cross the membranes of organelles directly and the reducing equivalents must be transported using shuttles, such as the chloroplast DHAP/3-PGA translocator mentioned above, or via the malate/oxaloacetate (Mal/OAA) shuttle [8] (Fig. 2). In chloroplasts, malate dehydrogenase (MDH) is NADP+-dependent, whereas an NAD+-MDH operates in the cytosol and the mitochondria. Chloroplast NADP+-MDH is activated in the light and converts OAA to malate when the chloroplast NADPH/NADP+ ratio is high [8, 9].
NAD(P)H 不能直接穿过细胞器的膜,还原当量必须使用穿梭机运输,例如上面提到的叶绿体 DHAP/3-PGA 转运机,或通过苹果酸/草酰乙酸 (Mal/OAA) 穿梭机运输 [8](图 2)。在叶绿体中,苹果酸脱氢酶 (MDH) 是 NADP+ 依赖性的,而 NAD+-MDH 在胞质溶胶和线粒体中起作用。当叶绿体 NADPH/NADP+ 比率较高时,叶绿体 NADP+-MDH 在光下被激活并将 OAA 转化为苹果酸 [89]。
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Fig. 2. Exchanges of reducing equivalents among chloroplasts, the cytosol and mitochondria. ETC, electron transport chain; GP, NADP+-GAPDH (glyceraldehyde-3-P dehydrogenase); NDX, externally facing NADPH dehydrogenase; NR, nitrate reductase.
图 2.叶绿体、胞质溶胶和线粒体之间还原当量的交换。ETC, 电子传递链;GP、NADP+-GAPDH(甘油醛-3-P 脱氢酶);NDX,面向外部的 NADPH 脱氢酶;NR,硝酸还原酶。

Mitochondria can also export reducing equivalents by exchanging citrate for cytosolic malate [1] (Fig. 3). Subsequent decarboxylation of citrate to 2-OG results in the production of NADPH. Reducing equivalents can also be exchanged across the chloroplast and mitochondrial membranes via the malate/aspartate shuttle, involving the malate/2-OG and glutamate/aspartate translocators [6]. However, the contribution of these two translocators to the transport of reducing equivalents is minor compared with the Mal/OAA shuttle [10].
线粒体还可以通过将柠檬酸盐交换为胞质苹果酸盐来输出还原当量 [1](图 3)。随后柠檬酸盐脱羧为 2-OG 导致 NADPH 的产生。还原当量也可以通过苹果酸/天冬氨酸穿梭子跨叶绿体和线粒体膜交换,涉及苹果酸/2-OG 和谷氨酸/天冬氨酸转运蛋白 [6]。然而,与 Mal/OAA 穿梭机相比,这两种转运器对还原当量运输的贡献很小 [10]。
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Fig. 3. Carbon exchange among chloroplasts, the cytosol and mitochondria. CS, citrate synthase; GOGAT, glutamate oxoglutarate transaminase; GP, NADP+-GAPDH (glyceraldehyde-3-P dehydrogenase); GS, glutamine synthase; MDH, malate dehydrogenase; ME, malic enzyme; PDC, pyruvate dehydrogenase complex; PEPC, PEP carboxylase; PK, pyruvate kinase.
图 3.叶绿体、胞质溶胶和线粒体之间的碳交换。CS,柠檬酸合酶;GOGAT,谷氨酸氧代戊二酸转氨酶;GP、NADP+-GAPDH(甘油醛-3-P 脱氢酶);GS,谷氨酰胺合酶;MDH,苹果酸脱氢酶;ME, 苹果酸酶;PDC,丙酮酸脱氢酶复合物;PEPC,PEP 羧化酶;PK,丙酮酸激酶。

Plant mitochondria can oxidise cytosolic NAD(P)H directly via the mitochondrial electron transport chain (ETC) using the externally facing NAD(P)H dehydrogenases [11] (Fig. 2). However, given the low concentrations of NADH (0.3–1.2 μM) and NADPH (150 μM) in the cytosol under physiological conditions and the substrate affinities of the external NAD(P)H dehydrogenase (Km; 1.4 μM for NADH and 80 μM for NADPH) [6, 8, 10, 12], it is most likely that only NADPH is oxidised by these NAD(P)H dehydrogenases, and even then at a very low rate.
植物线粒体可以使用面向外部的 NAD(P)H 脱氢酶 [11] 通过线粒体电子传递链 (ETC) 直接氧化胞质 NAD(P)H[11](图 2)。然而,鉴于生理条件下胞质溶胶中 NADH (0.3–1.2 μM) 和 NADPH (150 μM) 的低浓度以及外部 NAD(P)H 脱氢酶的底物亲和力(Km;NADH 为 1.4 μM,NADPH 为 80 μM)[681012],很可能只有 NADPH 被这些 NAD(P)H 脱氢酶氧化,即使如此,氧化率也非常低。

2.3. Exchange of carbon compounds
2.3. 碳化合物的交换

In addition to transporting ATP/ADP and reducing equivalents, mitochondria and chloroplasts also exchange carbon compounds. Chloroplasts export carbon at a high rate via the phosphate translocator [3] (Fig. 3). Cytosolic Pi concentrations determine whether DHAP remains in the chloroplast (to be converted to starch) or is exported (to serve as a substrate for sucrose, malate and/or pyruvate synthesis [13]).
除了运输 ATP/ADP 和还原当量外,线粒体和叶绿体还交换碳化合物。叶绿体通过磷酸盐转运器以高速率输出碳 [3](图 3)。胞质 P 浓度决定了 DHAP 是保留在叶绿体中(转化为淀粉)还是输出(作为蔗糖、苹果酸和/或丙酮酸合成的底物 [13])。
Mitochondria have specific organic acid translocators for most of the TCA cycle intermediates [14]. In addition to the Mal/OAA and the malate/citrate shuttles described in Section 2.2, malate can enter mitochondria via a dicarboxylate carrier which catalyses malate/Pi exchange [15].
线粒体对大多数 TCA 循环中间体具有特异性的有机酸转运体 [14]。除了第2.2节中描述的Mal/OAA和苹果酸盐/柠檬酸盐穿梭物外,苹果酸还可以通过催化苹果酸/P交换的二羧酸盐载体进入线粒体[15]。
In addition to being a reducing equivalent exchange system, the malate/citrate shuttle also exports carbon from the mitochondria (see Section 2.2). The 2-OG produced from cytosolic citrate decarboxylation serves as a carbon skeleton for amino acid synthesis in the chloroplast (Fig. 3). Import of 2-OG into the chloroplast is via the 2-OG/dicarboxylate exchange carrier that exchanges 2-OG for OAA and malate [14]. Whenever citrate is exported from the mitochondria, OAA or malate must be imported to replace the carbon lost from the TCA cycle. Because plant mitochondria have an NAD+-malic enzyme (NAD-ME) that converts malate to pyruvate, any TCA-cycle intermediate will suffice.
除了是一种还原等效交换系统外,苹果酸盐/柠檬酸盐穿梭机还从线粒体输出碳(参见第 2.2 节)。胞质柠檬酸脱羧产生的 2-OG 用作叶绿体中氨基酸合成的碳骨架(图 3)。2-OG 进入叶绿体是通过 2-OG/二羧酸盐交换载体将 2-OG 交换为 OAA 和苹果酸盐 [14]。每当柠檬酸盐从线粒体输出时,都必须进口 OAA 或苹果酸盐以补充 TCA 循环中损失的碳。因为植物线粒体具有将苹果酸转化为丙酮酸的 NAD+-苹果酸酶 (NAD-ME),所以任何 TCA 循环中间体都足够了。
The above sections demonstrate that plant cells possess a large number of transport systems to exchange metabolites among organelles and cytosol. The large array of transport systems help provide the cell with the metabolic flexibility it needs to respond to different conditions. However, the same variety of transport systems makes it very difficult to study the effects of altered conditions within the cell.
以上部分表明,植物细胞具有大量的运输系统,可在细胞器和胞质溶胶之间交换代谢物。大量的运输系统有助于为细胞提供应对不同条件所需的代谢灵活性。然而,相同种类的运输系统使得研究细胞内条件改变的影响变得非常困难。

3. Respiration in the light
3. 光下的呼吸

3.1. Does respiration continue in the light?
3.1. 呼吸在光中继续吗?

A question that has stimulated considerable debate is whether respiration continues in the light in photosynthetic cells, and, if so, whether it has the same rate as it does in the dark. Respiration (i.e. oxidative degradation of stored and recently fixed carbohydrates) is the main source of ATP for photosynthetic cells in the dark. In the past it was believed that respiration was fully inhibited in the light, probably as a result of photosynthetic ATP production, via adenylate control of glycolysis and limitations in substrate supply to the mitochondria [16]. This view is now considered too simplistic and experimental data suggest that mitochondrial activity continues in the light under most conditions. Mitochondria provide the cell with TCA cycle carbon skeletons for light-dependent NH+4 assimilation in the chloroplast (Fig. 3) and ATP and NADH for other biosynthetic reactions in the light (Fig. 1, Fig. 2). Mitochondria may also oxidise excess photosynthetic reducing equivalents in the light. Respiration is therefore likely to continue in the light, with the actual role that mitochondria serve in the light being dependent on the developmental stage and the environmental conditions.
一个引发了相当多争论的问题是,光合细胞中的呼吸是否在光照下继续存在,如果是这样,它是否与在黑暗中具有相同的速率。呼吸作用(即储存的和最近固定的碳水化合物的氧化降解)是黑暗中光合细胞 ATP 的主要来源。过去,人们认为呼吸在光线下被完全抑制,这可能是由于腺苷酸对糖酵解的控制和线粒体底物供应的限制而产生光合ATP [16]。这种观点现在被认为过于简单化,实验数据表明,在大多数情况下,线粒体活性在光线下继续存在。线粒体为细胞提供 TCA 循环碳骨架,用于叶绿体中光依赖性 NH+4 同化(图 3),并为 ATP 和 NADH 在光下进行其他生物合成反应(图 1图 2)。线粒体也可能在光中氧化过量的光合还原当量。因此,呼吸很可能在光下继续,线粒体在光中发挥的实际作用取决于发育阶段和环境条件。

3.2. Substrates for the mitochondria in the light
3.2. 光中线粒体的底物

Several substrates support respiration in the light, including photorespiratory glycine and products of recent photosynthetic activity, such as malate, OAA, pyruvate and NAD(P)H. Pärnik and Keerberg [17] defined these substrates as the primary products of photosynthesis. The degree to which primary products provide substrates for respiration is likely to increase under conditions where there is an excess of photosynthetic reducing equivalents (see Section 3.5.5).
几种底物支持光下的呼吸作用,包括光呼吸甘氨酸和近期光合活性的产物,如苹果酸、OAA、丙酮酸和 NAD(P)H。Pärnik 和 Keerberg [17] 将这些底物定义为光合作用的主要产物。在光合还原当量过量的条件下,初级产品为呼吸提供底物的程度可能会增加(参见第 3.5.5 节)。
Respiration of stored substrates (e.g. starch and sucrose) represents 40–50% of the total substrate oxidised by mitochondria in the light [17, 18] and 100% in the dark.
储存的底物(如淀粉和蔗糖)的呼吸作用在光下占线粒体氧化总底物的 40-50% [1718],在黑暗中占 100%。

3.3. ATP supply in the light: chloroplasts versus mitochondria
3.3. 光中的 ATP 供应:叶绿体与线粒体

The degree to which mitochondrial ATP supply in the light is required for optimal photosynthesis depends on the balance of ATP production and consumption in chloroplasts. It is possible that non-cyclic photosynthetic electron transport (Fig. 4), which produces ATP and NADPH in a ratio of 2.6:2 [19], does not satisfy the requirements of CO2 fixation and of other ATP-demanding processes in the chloroplast. Fixation of CO2 to yield DHAP in the chloroplast requires an ATP to NADPH ratio of 3:2 or greater. Additional ATP, if required, must therefore be provided to fix CO2 [20, 21] and for other cellular processes, such as sucrose synthesis, protein synthesis, NH+4 assimilation, metabolite transport and maintenance. Clearly, the demand for ATP can exceed the level of ATP synthesis by non-cyclic electron transport in the chloroplast, and additional ATP must be produced by other processes.
最佳光合作用需要光中线粒体 ATP 供应的程度取决于叶绿体中 ATP 产生和消耗的平衡。非循环光合电子传递(图 4)以 2.6:2 的比例产生 ATP 和 NADPH [19],可能无法满足叶绿体中 CO2 固定和其他 ATP 要求过程的要求。固定 CO2 以在叶绿体中产生 DHAP 需要 ATP 与 NADPH 的比率为 3:2 或更高。因此,如果需要,必须提供额外的ATP来固定CO2 [2021]和其他细胞过程,如蔗糖合成、蛋白质合成、NH+4同化、代谢物转运和维持。显然,对 ATP 的需求可能超过叶绿体中通过非环电子传递合成 ATP 的水平,并且必须通过其他过程产生额外的 ATP。
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Fig. 4. Organisation of the thylakoid membrane. FNR, ferredoxin-NADP+ oxidoreductase; FQR, ferredoxin-plastoquinone reductase; LHC, light harvesting complex; MR, Mehler reaction; PC, plastocyanin; NAD(P)HDH, NAD(P)H dehydrogenase; PQ, plastoquinone; PS, photosystem. The elements of the suggested chlororespiratory pathway are indicated by dark-shaded rectangles.
图 4.类囊体膜的组织。FNR,铁氧还蛋白-NADP+ 氧化还原酶;FQR,铁氧还蛋白-质体醌还原酶;LHC,光收集复合物;MR,Mehler 反应;PC,质体蓝蛋白;NAD(P)HDH、NAD(P)H 脱氢酶;PQ,质体醌;PS,光系统。建议的叶子呼吸途径的元素由深色矩形表示。

The degree to which mitochondria provide ATP to the chloroplast depends on the contribution from cyclic [22, 23] and pseudo-cyclic [22, 24] phosphorylation (Fig. 4). In cyclic phosphorylation, the acceptor of PSI (Fd or NADPH) is oxidised by PQ, which serves as a donor to PSI, yielding ATP as the sole product. Experiments with pea leaves suggested that substantial cyclic phosphorylation will only occur at high irradiances in combination with very low CO2 concentrations [25]. In pseudo-cyclic phosphorylation oxidation of the PSI acceptor produces H2O2 (Fig. 4), which is rapidly removed by catalase, and depends on both PSI and PSII. Although these two processes have sufficient capacity to meet the demand for extra ATP [23, 24], they probably play a minor role in vivo [6]. However, little additional ATP synthesis may be needed to balance the NADPH:ATP ratio, allowing the Calvin cycle to operate.
线粒体向叶绿体提供 ATP 的程度取决于环状 [2223] 和伪环状 [2224] 磷酸化的贡献(图 4)。在环磷酸化中,PSI 的受体(Fd 或 NADPH)被 PQ 氧化,PQ 作为 PSI 的供体,产生 ATP 作为唯一的产物。对豌豆叶片的实验表明,只有在高辐照度和极低的 CO2 浓度下,才会发生大量的循环磷酸化 [25]。在伪环磷酸化中,PSI 受体的氧化产生 H2O2图 4),它被过氧化氢酶迅速去除,并且依赖于 PSI 和 PSII。尽管这两个过程有足够的能力来满足对额外ATP的需求[23,24],但它们在体内可能起着次要的作用[6]。然而,可能只需要很少的额外 ATP 合成来平衡 NADPH:ATP 比率,从而允许卡尔文循环运行。
If the chloroplast is unable to meet its ATP requirements, additional ATP must be imported from other compartments of the cell. The most likely source of additional ATP is mitochondrial phosphorylation. Mitochondria have a greater capacity for ATP synthesis than chloroplasts, producing up to 3 ATP per NAD(P)H compared to the 1.5–2 ATP per NAD(P)H in the chloroplast [26]. Indeed, mitochondrial oxidative phosphorylation maintains most of the cytosolic ATP pool [6] and is essential for maximal rates of tissue photosynthesis in some instances [27, 28, 29, 30]. Experiments with barley leaf protoplasts showed that photosynthetic O2 evolution was 30–40% lower when mitochondrial ATP production was inhibited by oligomycin at a concentration that did not affect the process of photosynthesis directly [30]. Subsequent rupturing of the protoplasts that left the chloroplasts intact restored the photosynthetic rate [30]. These experiments suggest that under these conditions mitochondrial ATP production was essential for optimal photosynthesis and may reflect the energy demands of sucrose synthesis, which utilises UTP [6, 27] (Fig. 1).
如果叶绿体无法满足其 ATP 要求,则必须从细胞的其他隔室导入额外的 ATP。额外 ATP 最可能的来源是线粒体磷酸化。线粒体比叶绿体具有更强的ATP合成能力,每NAD(P)H可产生多达3个ATP,而叶绿体中每NAD(P)H可产生1.5-2个ATP[26]。事实上,线粒体氧化磷酸化维持了大部分胞质 ATP 库 [6],并且在某些情况下对组织光合作用的最大速率至关重要 [27282930]。大麦叶原生质体的实验表明,当寡霉素以不直接影响光合作用过程的浓度抑制线粒体ATP的产生时,光合O2的释放量降低了30-40%[30]。随后原生质体破裂,叶绿体完好无损,恢复了光合速率[30]。这些实验表明,在这些条件下,线粒体 ATP 的产生对于最佳光合作用至关重要,并且可能反映了利用 UTP 的蔗糖合成的能量需求 [627](图 1)。
The degree to which mitochondrial ATP production is necessary for cell function in the light is likely to vary among tissues. For example, the amount of ATP produced in non-cyclic electron transport in the chloroplasts appears to be sufficient to account for CO2 uptake in photoautotrophic carnation cell cultures, without involving cyclic phosphorylation or mitochondrial ATP production [31]. However, mitochondria may still contribute to cellular ATP synthesis in such cells, for other energy demanding processes that occur in the light (e.g. N-assimilation). Environmental factors can also affect the need for mitochondria to supply ATP. For example, Hurry et al. [32] reported that mitochondria contribute to ATP pools in illuminated non-hardened leaves of winter rye, but not in cold-hardened leaves.
线粒体 ATP 产生对细胞在光线下功能的必要程度可能因组织而异。例如,叶绿体中非环状电子传递产生的ATP量似乎足以解释光合自养康乃馨细胞培养物中CO2的摄取,而不涉及循环磷酸化或线粒体ATP的产生[31]。然而,线粒体仍可能有助于此类细胞中的细胞 ATP 合成,用于在光下发生的其他需要能量的过程(例如 N 同化)。环境因素也会影响线粒体供应 ATP 的需求。例如,Hurry等[32]报道,线粒体有助于冬黑麦光照的非硬化叶片中的ATP池,但在寒冷硬化的叶片中则没有。

3.4. Adenylate control of respiration in the light
3.4. 腺苷酸在光下控制呼吸

Adenylates can restrict respiration in various ways [33]. Firstly, in isolated mitochondria an ATP/ADP ratio higher than 20 will restrict oxidative phosphorylation [34], a ratio reported to occur in vivo [35]. Secondly, phosphorylation can be restricted if the concentration of ADP is too low (below 20–50 μM; depending on the ATP/ADP ratio [34]). About 40–50% of cellular ADP is bound to proteins and in maize root tips the concentration of free ADP was estimated to be about 50 μM [36], within the concentration range where it restricts phosphorylation. Low concentrations of free ADP (as distinct from ATP/ADP ratios) may be more important in metabolic regulation than previously recognised. Thirdly, the rate of glycolysis is regulated by the concentrations of ATP and ADP in the cytosol: an increase in the ATP concentration will decrease the activity of key enzymes of glycolysis. Small increases in the ATP/ADP ratio in the cytosol are sufficient to modify the rate of glycolysis [37]. Moreover, low ADP concentrations can restrict the rate of substrate level phosphorylation, especially at pyruvate kinase (PK, Fig. 1) [38].
腺苷酸酯可以通过多种方式限制呼吸[33]。首先,在分离的线粒体中,高于 20 的 ATP/ADP 比率将限制氧化磷酸化 [34],据报道该比率发生在体内 [35]。其次,如果 ADP 浓度过低(低于 20–50 μM;取决于 ATP/ADP 比率 [34]),则可以限制磷酸化。大约 40-50% 的细胞 ADP 与蛋白质结合,在玉米根尖中,游离 ADP 的浓度估计约为 50 μM [36],在其限制磷酸化的浓度范围内。低浓度的游离 ADP(与 ATP/ADP 比率不同)在代谢调节中可能比以前认识到的更重要。第三,糖酵解速率受胞质溶胶中 ATP 和 ADP 浓度的调节:ATP 浓度的增加会降低糖酵解关键酶的活性。胞质溶胶中 ATP/ADP 比率的小幅增加足以改变糖酵解速率 [37]。此外,低 ADP 浓度会限制底物水平磷酸化的速率,尤其是在丙酮酸激酶处(PK,图 1)[38]。
Cytosolic ATP/ADP ratios in the light are similar or lower than in darkness [39, 40, 41], suggesting that respiration is not completely inhibited by adenylates in the light. However, the lower ATP/ADP ratios in the light than in darkness [41] may actually reflect a faster turnover of ATP in the light, rather than a lower ATP level per se. It is likely therefore, that respiration is restricted by adenylates in the light and the dark. Despite this, respiration generally continues in the light (see Section 3.8): the degree of adenylate control is insufficient to fully inhibit respiration in the light. Plant cells also have mechanisms for avoiding adenylate control; their mitochondria have non-phosphorylating pathways, allowing respiration without ATP production (see Section 3.5.6). Similarly, glycolytic adenylate control can be avoided if PEP is converted to malate by PEP carboxylase (PEPC), bypassing PK which can be limited by low ADP [42] (Fig. 3). Thus, plant metabolism need not be as strongly controlled by adenylates as animal metabolism, giving the plant cells greater flexibility.
光中的胞质 ATP/ADP 比率与黑暗中相似或更低 [394041],这表明光中的腺苷酸并未完全抑制呼吸。然而,光明下的 ATP/ADP 比率低于黑暗中 [41] 实际上可能反映了 ATP 在光明下的更快周转,而不是 ATP 水平本身较低。因此,呼吸很可能受到光明黑暗中的腺苷酸的限制。尽管如此,呼吸通常在光线下继续(参见第 3.8 节):腺苷酸控制程度不足以完全抑制光线下的呼吸。植物细胞还具有避免腺苷酸控制的机制;它们的线粒体具有非磷酸化途径,允许在不产生 ATP 的情况下进行呼吸(参见第 3.5.6 节)。同样,如果 PEP 被 PEP 转化为苹果酸,绕过可能受到低 ADP 限制的 PK,则可以避免糖酵解腺苷酸控制 [42](图 3)。因此,植物代谢不需要像动物代谢那样受到腺苷酸的强烈控制,从而赋予植物细胞更大的灵活性。

3.5. Mechanisms to avoid over-reduction of the chloroplast
3.5. 避免叶绿体过度还原的机制

Another role for mitochondria in the light may be the removal of excess photosynthetic reducing equivalents, which can lead to damage of the photosynthetic electron transport system. It is therefore essential that chloroplasts export excess reducing equivalents to be either stored (e.g. as malate) or to be oxidised by respiration.
线粒体在光下的另一个作用可能是去除多余的光合还原当量,这可能导致光合电子传输系统的损伤。因此,叶绿体必须输出过量的还原当量,以便储存(例如苹果酸盐)或通过呼吸作用氧化。

3.5.1. Over-reduction and photoinhibition
3.5.1. 过度还原和光抑制

When the chloroplast NADPH/NADP ratio becomes too high, photosynthetic electron transport components will become highly reduced, resulting in photoinhibition [43], which reduces photosynthetic efficiency [44] and occurs when the ability of the photosynthetic ETC to readily dissipate absorbed energy, either photochemically (e.g. fluorescence, ATP and NADPH synthesis) or non-photochemically (e.g. dissipation of energy as heat), is reduced. This results in a change or damage to the photosynthetic apparatus (mostly likely to the D1 protein of PSII [43]). Therefore, other pathways for dissipation of energy in PSII need to exist, e.g. fluorescence, state transitions and the xanthophyll cycle.
当叶绿体 NADPH/NADP 比率变得过高时,光合电子传输成分将高度减少,导致光抑制 [43],从而降低光合效率 [44],并且发生在光合 ETC 易于耗散吸收能量的能力时,无论是光化学的(例如荧光、ATP 和 NADPH 合成)还是非光化学的(例如能量作为热量的耗散), 减少。这会导致光合装置发生变化或受损(最有可能是PSII的D1蛋白[43])。因此,需要存在 PSII 中能量耗散的其他途径,例如荧光、状态转换和叶黄素循环。

3.5.2. State transitions and the xanthophyll cycle
3.5.2. 状态转换和叶黄素循环

In state transitions, light harvesting complexes (LHCs) move from one reaction centre to the reaction centre of the other photosystem (for a review see [45]). A highly reduced PQ pool induces a transition to state II, when LHCs of PSII are phosphorylated and move to PSI. A return to state I requires ATP and a highly oxidised PQ pool [46]. These transitions modulate flux through PSI and the rate of PQ oxidation, balancing the energy distribution between the two photosystems and avoiding over-reduction of the ETC components, especially of PQ. Over-reduction of quinone pools in mitochondria or chloroplasts can lead to the production of active oxygen species, which can damage the cell [47, 48, 49]. State transitions affect the degree of cyclic and non-cyclic phosphorylation and change the ratio of NADPH:ATP production.
在状态转变中,光捕获复合物 (LHC) 从一个反应中心移动到另一个光系统的反应中心(综述见 [45])。当 PSII 的 LHC 被磷酸化并转移到 PSI 时,高度还原的 PQ 池会诱导向状态 II 的转变。恢复到状态 I 需要 ATP 和高度氧化的 PQ 池 [46]。这些跃迁通过 PSI 调节通量和 PQ 氧化速率,平衡两个光系统之间的能量分配,并避免 ETC 分量的过度还原,尤其是 PQ。线粒体或叶绿体中醌库的过度减少会导致活性氧的产生,从而损害细胞[47,48,49]。 状态转变会影响环和非环磷酸化的程度,并改变 NADPH:ATP 产生的比率。
State transitions have a limited capacity to protect photosystems against photoinhibition, because they only re-distribute the photochemical energy between the photosystems and also PSI can become photoinhibited [50]. The xanthophyll cycle, on the other hand, protects both photosystems [50], allowing LHCs to dissipate energy as heat and reducing photo-efficiency [50, 51, 52]. The heat dissipation capacity of the xanthophyll cycle only increases when the plant is exposed to high light for a long time [52].
状态转换保护光系统免受光抑制的能力有限,因为它们只会在光系统之间重新分配光化学能,而且 PSI 也会受到光抑制 [50]。另一方面,叶黄素循环保护两个光系统[50],使LHC能够以热量的形式耗散能量并降低光效率[50,51,52]。 叶黄素循环的散热能力只有在植物长时间暴露在强光下时才会增加[52]。
The above systems do not provide complete protection against photoinhibition. They are also only invoked when the ETC is already highly reduced (e.g. state transitions) or when the plant has been exposed to photoinhibitory light for an extended periods (e.g. xanthophyll cycle). The xanthophyll cycle cannot dissipate all excess photochemical energy under stress conditions [50]. Further, these protective mechanisms reduce photosynthetic efficiency. It would therefore be beneficial to have other systems to deal with dissipation of excess chloroplast energy, especially for short-term transient imbalances.
上述系统不能提供针对光抑制的完全保护。它们也仅在 ETC 已经高度减少(例如状态转换)或植物长时间暴露在光抑制光下(例如叶黄素循环)时调用。在胁迫条件下,叶黄素循环不能消散所有多余的光化学能[50]。此外,这些保护机制降低了光合作用效率。因此,拥有其他系统来处理过量叶绿体能量的耗散将是有益的,特别是对于短期瞬态不平衡。

3.5.3. Avoiding over-reduction: sinks for NADPH and ATP
3.5.3. 避免过度还原:NADPH 和 ATP 的汇

Imbalances leading to over-reduction of the ETC typically occur when the supply of NADPH and ATP exceeds the demand for these metabolites. The electron flow in the chloroplast ETC can be limited by a low availability of NADP+ (terminal acceptor) or ADP. Because electron transport is coupled to ATP synthesis it is restricted in the absence of ADP (similar to the ‘state 4’ of mitochondria) [53]. Therefore, regeneration of ADP is also important for unobstructed photosynthetic electron flow. While chloroplasts have mechanisms to produce ATP without NADPH, there is no photosynthetic system to produce NADPH without ATP. However, given the ratio in which NADPH and ATP are produced, NADPH is generally in excess (see Section 3.5.1). Over-reduction can be avoided if the rate of NADPH and ATP production is matched or exceeded by the potential consumption of these metabolites and/or if excess metabolites are exported from the chloroplast.
当 NADPH 和 ATP 的供应超过对这些代谢物的需求时,通常发生导致 ETC 过度减少的失衡。叶绿体 ETC 中的电子流可能受到 NADP+(末端受体)或 ADP 可用性低的限制。由于电子传递与 ATP 合成耦合,因此在没有 ADP 的情况下(类似于线粒体的“状态 4”)会受到限制 [53]。因此,ADP 的再生对于畅通无阻的光合电子流也很重要。虽然叶绿体具有在没有 NADPH 的情况下产生 ATP 的机制,但没有 ATP 就没有光合系统来产生 NADPH。然而,考虑到 NADPH 和 ATP 的产生比例,NADPH 通常是过量的(参见第 3.5.1 节)。如果 NADPH 和 ATP 的产生速率与这些代谢物的潜在消耗相匹配或超过和/或如果过量的代谢物从叶绿体中输出,则可以避免过度还原。
Photosynthetic CO2 fixation and photorespiration (Fig. 5) require substantial amounts of NADPH and ATP. CO2 and O2 compete for binding sites on Rubisco, with 20–35% of the net photosynthetic activity occurring by the oxygenase reaction (photorespiration) under normal conditions [54, 55]. In the Calvin cycle two 3-PGA are produced for each RuBP, whereas photorespiration results in the conversion of RuBP to 3-PGA and 2-P-glycolate (Fig. 5). The carbon lost to glycolate is salvaged in the photorespiratory cycle with the evolution of CO2 and NH3 (Fig. 5; for details see [56]). 2-P-Glycolate is converted to glycolate and exported to the peroxisome, where the glycolate is converted to glycine and then metabolised in the mitochondria as a respiratory substrate. The photorespiratory glycolate cycle provides a substantial sink for NADPH and ATP (2 NADPH and 3.5 ATP per glycolate; totalling 4 NADPH and 6.5 ATP per oxygenation if the re-fixation of lost CO2 is included), especially under conditions when the carboxylation reaction is limited by low intercellular CO2 concentrations (e.g. following stomatal closure).
光合 CO2 固定和光呼吸(图 5)需要大量的 NADPH 和 ATP。CO2 和 O2 在 Rubisco 上竞争结合位点,在正常条件下,20-35% 的净光合活性由加氧酶反应(光呼吸)发生 [5455]。在卡尔文循环中,每个 RuBP 产生两个 3-PGA,而光呼吸导致 RuBP 转化为 3-PGA 和 2-P-乙醇酸盐(图 5)。乙醇酸盐损失的碳在光呼吸循环中随着 CO2 和 NH3 的释放而被挽救(图 5;详情见 [56])。2-P-乙醇酸盐转化为乙醇酸盐并输出到过氧化物酶体,在那里乙醇酸盐转化为甘氨酸,然后作为呼吸底物在线粒体中代谢。光呼吸乙醇酸盐循环为 NADPH 和 ATP 提供了相当大的汇(每个乙醇酸盐 2 个 NADPH 和 3.5 个 ATP;如果包括丢失的 CO2 的重新固定,则每次氧合总计 4 个 NADPH 和 6.5 个 ATP),尤其是在羧化反应受低细胞间 CO2 浓度限制的条件下(例如,气孔关闭后)。
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Fig. 5. Photorespiration or glycolate cycle. GOGAT, glutamate oxoglutarate transaminase; GS, glutamine synthase.
图 5.光呼吸或乙醇酸盐循环。GOGAT,谷氨酸氧代戊二酸转氨酶;GS,谷氨酰胺合酶。

Because CO2 fixation is such an important sink for chloroplast NADPH and ATP, it must be active or rapidly activated whenever photons are absorbed if the chloroplast is to avoid over-reduction. In darkness, chloroplastic enzymes that use NADPH (e.g. Calvin cycle) are typically inactive. The redox state of the chloroplast increases dramatically during dark to light transitions, with Fd, Td and NADPH levels increasing as a result of photosynthetic electron flow [57, 58]. This could result in a build up of excess NADPH and over-reduction of the ETC and rapid activation of the processes that use photosynthetic NADPH is therefore needed. Indeed, enzymes of the Calvin cycle (e.g. NADP+-GAPDH, fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase and phosphoribulokinase (PRK)) are rapidly activated in the light, by the increased Td levels [9, 57], which reduce a sulphydryl group oxidised by O2. The light-regulated Calvin cycle enzymes are continually reduced and oxidised ensuring that their activity is tightly controlled, with overall regulation being controlled by the redox state of the chloroplast.
因为 CO2 固定是叶绿体 NADPH 和 ATP 的重要汇,所以如果要避免叶绿体过度还原,只要光子被吸收,它就必须活跃或迅速激活。在黑暗中,使用 NADPH(例如卡尔文循环)的叶绿酶通常是无活性的。在从暗到亮的转变过程中,叶绿体的氧化还原状态急剧增加,Fd、Td和NADPH水平由于光合电子流而增加[57,58]。 这可能导致过量 NADPH 的积累和 ETC 的过度减少,因此需要快速激活使用光合 NADPH 的过程。事实上,卡尔文循环的酶(例如 NADP+-GAPDH、果糖 1,6-二磷酸酶、景天庚糖 1,7-二磷酸酶和磷酸盐激酶 (PRK))在光线下被升高的 Td 水平迅速激活 [957],从而减少被 O2 氧化的巯基。光调节的卡尔文循环酶不断被还原和氧化,确保它们的活性受到严格控制,整体调节由叶绿体的氧化还原状态控制。
In addition to carbon fixation and photorespiration, another important sink for NADPH and ATP is nitrogen assimilation. NADPH exported from the chloroplast can be used for the cytosolic reduction of NO3 to NO2 by nitrate reductase (NR; Fig. 2), which is inactivated within minutes in the dark [59]. In the chloroplast, NO2 is converted to NH+4 using reduced Fd. The rate of NO3 assimilation is typically about 4% of CO2 fixation and uses 10% of the reducing equivalents used for CO2 fixation [60]. However, this value will vary substantially between species, developmental stages and environmental conditions. Limitations in NO3 supply, in particular, will influence the rate of nitrogen assimilation and thus the demand for reducing equivalents in illuminated leaves. Similarly, the demands for ATP associated with nitrogen assimilation will vary as a function of the rate of nitrogen assimilation: substantial amounts of ATP are needed for NH+4 assimilation and amino acid synthesis [61]. Clearly, nitrogen assimilation provides a major sink for chloroplast NADPH and ATP. High rates of nitrogen assimilation should, therefore, reduce the potential for over-reduction of the photosynthetic ETC.
除了碳固定和光呼吸之外,NADPH 和 ATP 的另一个重要汇是氮同化。从叶绿体输出的 NADPH 可用于通过硝酸盐还原酶 (NR;图 2),它在黑暗中在几分钟内失活 [59]。在叶绿体中,NO-2 使用还原的 Fd 转化为 NH + 4NO-3 的同化速率通常约为 CO2 固定的 4%,使用用于 CO2 固定的还原当量的 10% [60]。然而,该值会因物种、发育阶段和环境条件而异。特别是 NO3 供应的限制会影响氮的同化速率,从而影响对照明叶中减少当量的需求。同样,与氮同化相关的 ATP 需求将随氮同化速率而变化:NH+4 同化和氨基酸合成需要大量的 ATP [61]。显然,氮同化为叶绿体 NADPH 和 ATP 提供了主要汇。因此,高氮同化率应减少光合 ETC 过度还原的可能性。
The effects of nitrogen assimilation on photosynthesis and respiration have been studied extensively in green algae (for a review see [61]). Addition of NO3 or NH+4 to nitrogen-starved algae diverts the flow of photosynthetic electrons away from CO2 fixation to nitrogen assimilation [62], lowering the level of reduction of the chloroplast, and reducing the activity of the CO2 fixing enzymes (e.g. phosphoribulose kinase and G6P-dehydrogenase [63, 64]). When NH+4 is added instead of NO3 (thus lowering the demand for reducing equivalents for nitrogen assimilation), PRK is not inhibited, demonstrating the strong redox regulation of this process. The slowdown of PRK upon NO3 addition inhibits the regeneration phase of the reductive pentose phosphate pathway and leads to an increase in RuBP and a decrease in photosynthesis. Experiments with isolated spinach chloroplasts have also demonstrated that NO3 reduction lowers the rate of photosynthesis, due to the diversion of reductant from CO2 fixation to nitrogen assimilation [65]. However, photosynthesis is unlikely to be limited by the NADPH demand of nitrogen assimilation very often, as electron flow in the chloroplasts is frequently in excess of that required for CO2 fixation and photorespiration [66, 67].
氮同化对光合作用和呼吸作用的影响已在绿藻中得到广泛研究(综述见 [61])。向缺氮藻类中添加NO-3或NH+4会使光合电子的流动从CO2固定转移到氮同化[62],降低叶绿体的还原水平,并降低CO2固定酶(如磷酸-布洛糖激酶和G6P-脱氢酶[63,64])的活性。 当添加 NH+4 而不是 NO-3 时(从而降低了氮同化对还原当量的需求),PRK 不会受到抑制,表明该过程具有很强的氧化还原调节。添加 NO-3 后 PRK 的减慢抑制了还原性磷酸戊糖途径的再生阶段,并导致 RuBP 增加和光合作用减少。对分离的菠菜叶绿体的实验也表明,由于还原剂从 CO2 固定转移到氮同化,NO-3 还原降低了光合作用速率 [65]。然而,光合作用不太可能经常受到 NADPH 氮同化需求的限制,因为叶绿体中的电子流经常超过固定 CO2 和光呼吸所需的电子流 [6667]。

3.5.4. Avoiding over-reduction: export of excess NADPH via the Mal/OAA shuttle
3.5.4. 避免过度减少:通过 Mal/OAA 穿梭车输出超额 NADPH

Over-reduction of the chloroplast can also be avoided via export of excess reducing equivalents to other cell compartments. The primary export mechanism appears to be the Mal/OAA shuttle mechanism described in Section 2.2: NADPH reduces OAA to malate (via NADP+-MDH), which is exported from the chloroplast (Fig. 3). NADP+-MDH is activated by high NADPH levels in the chloroplast and this activation is inhibited by O2 and NADP+ [9].
也可以通过将过量的还原当量输出到其他细胞区室来避免叶绿体的过度还原。主要的输出机制似乎是第 2.2 节中描述的 Mal/OAA 穿梭机制:NADPH 将 OAA 还原为苹果酸(通过 NADP+-MDH),苹果酸从叶绿体输出(图 3)。NADP+-MDH 被叶绿体中高水平的 NADPH 激活,这种激活被 O2 和 NADP+ 抑制 [9]。
In the absence of OAA, NADPH to can also be re-oxidised in the chloroplast by the Mehler reaction, consuming O2 (Fig. 4), which has been suggested to be an alternative Hill oxidant acting as a fail/safe system [24]. However, the Mal/OAA shuttle appears to be preferred, because H2O2 production stops when OAA is added to illuminated chloroplasts [68]. Experiments with spinach and sunflower leaves showed that the Mehler reaction is not sufficient to protect against photoinactivation [69].
在没有 OAA 的情况下,NADPH 也可以在叶绿体中通过 Mehler 反应重新氧化,消耗 O2图 4),这被认为是一种替代的 Hill 氧化剂,作为故障/安全系统[24]。然而,Mal/OAA穿梭似乎是首选,因为当OAA加入到发光的叶绿体中时,H2O2的产生会停止[68]。对菠菜和葵花籽叶的实验表明,Mehler 反应不足以防止光灭活 [69]。

3.5.5. Oxidation of excess photosynthetic reductant outside the chloroplast
3.5.5. 叶绿体外过量光合还原剂的氧化

For the Mal/OAA shuttle to operate as an effective NADPH export system, the exported malate must be oxidised to regenerate OAA for transport back to the chloroplast (Fig. 3). Malate can be oxidised in the cytosol, peroxisomes or the mitochondria, using the reducing equivalents for various reactions, such as NO3 reduction in the cytosol or reduction of hydroxypyruvate in the peroxisomes. Under conditions where more reductant is produced than is required for cytosolic and peroxisome processes, malate can be imported into the mitochondria for oxidation. Experimental evidence indicates that mitochondrial activity in the light can reduce photoinhibition and that this protection is probably related to the removal of excess photosynthetic reducing equivalents [32, 70, 71, 72, 73].
为了使 Mal/OAA 穿梭车作为有效的 NADPH 输出系统运行,输出的苹果酸必须被氧化以再生 OAA 以运输回叶绿体(图 3)。苹果酸可以在胞质溶胶、过氧化物酶体或线粒体中被氧化,使用还原当量进行各种反应,例如胞质溶胶中的 NO-3 还原或过氧化物酶体中羟基丙酮酸的还原。在产生的还原剂多于胞质和过氧化物酶体过程所需的还原剂的情况下,苹果酸可以输入到线粒体中进行氧化。实验证据表明,光中的线粒体活性可以减少光抑制,这种保护可能与去除多余的光合作用还原当量有关[32,70,71,72,73]。
A recent study has suggested that proline synthesis may be another way of re-oxidising excess NAD(P)H in the cell [74]. Proline has long been recognised as a metabolite that accumulates during stress and is rapidly oxidised once the stress is removed. It may be that the ATP produced during its oxidation is important in the recovery from stress.
最近的一项研究表明,脯氨酸合成可能是重新氧化细胞中过量 NAD(P)H 的另一种方式 [74]。长期以来,脯氨酸一直被认为是一种代谢物,在应激期间积累,一旦应激被消除,就会迅速氧化。氧化过程中产生的 ATP 对于从压力中恢复很重要。

3.5.6. Role of non-phosphorylating pathways in avoiding over-reduction
3.5.6. 非磷酸化途径在避免过度还原中的作用

Oxidation of reducing equivalents in the mitochondria can be coupled to the production of ATP. Under conditions where ATP demand is low, the recycling of ADP would limit the rate of oxidation. However, the existence of non-phosphorylating bypasses in the ETC of plant mitochondria allows electron flow to continue even when the demand for ATP is limited and the ADP availability is low [75]. These include the alternative oxidase (AOX) [76], a quinol oxidase with O2 as its acceptor that bypasses complexes III and IV in the mitochondrial ETC (Fig. 6). Plant mitochondria also have non-phosphorylating NADH dehydrogenases that bypass complex I [11] and can oxidise internal and external NADH without any ATP production and without any requirement for ADP (Fig. 6).
线粒体中还原当量的氧化可以与 ATP 的产生偶联。在 ATP 需求较低的条件下,ADP 的回收将限制氧化速率。然而,植物线粒体的 ETC 中存在非磷酸化旁路,即使在对 ATP 的需求有限且 ADP 可用性较低的情况下,电子也可以继续流动 [75]。这些包括替代氧化酶 (AOX) [76],这是一种以 O2 为受体的喹醇氧化酶,可绕过线粒体 ETC 中的复合物 III 和 IV(图 6)。植物线粒体还具有绕过复合物 I 的非磷酸化 NADH 脱氢酶 [11],可以氧化内部和外部 NADH,而无需产生任何 ATP,也不需要任何 ADP(图 6)。
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Fig. 6. Organisation of the plant mitochondrial membrane. AOX, alternative oxidase; NDRI, matrix-side rotenone insensitive NADH dehydrogenase; NDX, external NAD(P)H dehydrogenase(s); SDH, succinate dehydrogenase. The non-phosphorylating bypasses are dark-shaded.
图 6.植物线粒体膜的组织。AOX,替代氧化酶;NDRI,基质侧鱼藤酮不敏感的 NADH 脱氢酶;NDX,外部 NAD(P)H 脱氢酶;SDH,琥珀酸脱氢酶。非磷酸化旁路为深色阴影。

Taken together, the above discussion demonstrates that photosynthetic cells have a diverse range of systems to deal with excess reducing equivalents that gives them flexibility to respond to various conditions.
综上所述,上述讨论表明,光合电池具有多种系统来处理过量的还原当量,这使它们能够灵活地响应各种条件。

3.6. Environmental factors and excess NADPH
3.6. 环境因素和过量的 NADPH

The imbalance between the production and consumption of NADPH and ATP will be accentuated under adverse environmental conditions, such as when the demand for NADPH and ATP for biosynthesis is limited (e.g. by low temperatures or nutrient limitations) or when the ability to use these metabolites for CO2 fixation is restricted by low internal CO2 concentrations (e.g. during drought). Under those conditions the rate of processes involved in removing excess NAD(P)H will increase.
在不利的环境条件下,NADPH 和 ATP 的生产和消费之间的不平衡将加剧,例如当生物合成对 NADPH 和 ATP 的需求受到限制时(例如由于低温或营养限制),或者当使用这些代谢物固定 CO2 的能力受到内部 CO2 浓度的限制时(例如在干旱期间)。在这些条件下,去除过量 NAD(P)H 所涉及的过程速率将增加。

3.6.1. Excess photosynthetic reductant: low temperatures and high irradiance
3.6.1. 过量的光合还原剂:低温和高辐照度

It is well known that low temperatures increase the susceptibility of plants to photoinhibition. At low temperatures (e.g. less than 10°C for plants growing in moderate climates) sucrose synthesis is severely limited. This restricts the recycling of Pi and the export of DHAP, inhibiting the Calvin cycle and the use of photosynthetic NADPH [77] (Fig. 1). Therefore, plants are much more susceptible to photoinhibition under cold conditions even at moderate light intensities. The deleterious effects of bright light and cold temperatures may, however, be ameliorated by the oxidation of excess photosynthetic reducing equivalents by the mitochondria [32, 71, 72]. Respiratory rates at a given temperature also increase in plants that are exposed to cold temperatures for extended periods [32, 78]: the increase in respiratory capacity may represent an increased capacity to oxidise excess photosynthetic reducing equivalents. Plants also acclimate to low temperatures by increasing photosynthetic and sucrose synthesis activity [32, 79], and reducing the Pi-mediated feedback inhibition of photosynthesis [77].
众所周知,低温会增加植物对光抑制的敏感性。在低温下(例如,在温和气候下生长的植物低于 10°C),蔗糖合成受到严重限制。这限制了 P 的循环和 DHAP 的出口,抑制了卡尔文循环和光合 NADPH 的使用[77](图 1)。因此,即使在中等光照强度下,植物在寒冷条件下也更容易受到光抑制。然而,强光和低温的有害影响可以通过线粒体氧化过量的光合还原当量来减轻[32,71,72]。在给定温度下,长时间暴露在低温下的植物的呼吸频率也会增加[32,78]:呼吸能力的增加可能代表氧化过量光合作用还原当量的能力增加。植物还通过增加光合作用和蔗糖合成活性[32,79]和减少P介导的光合作用反馈抑制来适应低温[77]。

3.6.2. Excess photosynthetic reductant: low intercellular CO2 and drought
3.6.2. 过量的光合还原剂:低细胞间 CO2 和干旱

Severe inhibition of photosynthesis can be expected when intercellular CO2 concentrations (ci) are low (as occurs when stomata close), as CO2 fixation provides the largest sink for photosynthetic NADPH and ATP. Photoinhibition is enhanced in Phaseolus vulgaris leaves when ci is reduced [80]. Increases in ci also result in an increased rate of CO2 fixation and a decrease in the ATP/ADP ratio in spinach leaves [81]. Low ci values therefore reduce the demand for NADPH and ATP, and increase the potential for photoinhibition.
当细胞间 CO2 浓度 (c) 较低时(如气孔关闭时发生),可以预期光合作用会受到严重抑制,因为 CO2 固定为光合 NADPH 和 ATP 提供了最大的汇。当 c 降低时,豆叶片的光抑制增强 [80]。c 的增加也会导致菠菜叶片中 CO2 固定速率的增加和 ATP/ADP 比率的降低 [81]。因此,低 c 值减少了对 NADPH 和 ATP 的需求,并增加了光抑制的可能性。
Although stomatal closure and low ci values decrease CO2 fixation rates, they do not reduce the rate of photorespiration. In fact, it is slightly increased at low ci values [82] and with this the demand for NADPH and ATP for photorespiration is maintained or increased. Photorespiration thus helps avoid over-reduction of the ETC and long-term damage to the photosystem under conditions where CO2 fixation is limited by low ci values [20, 43]. This is likely to be particularly important under drought conditions when stomata are closed. Various studies have shown that photorespiration increases during drought and offers protection against photoinhibition [20, 80, 82, 83, 84]. In Digitalis lanata water stress reduces net photosynthesis by 70%; however, the metabolic demand for energy decreases only 40% due to continued demand for NADPH and ATP by photorespiration and because much of the CO2 released by mitochondrial glycine decarboxylation is reassimilated by Rubisco in the chloroplast [82]. By maintaining the demand for these metabolites, D. lanata is able to avoid over-reduction of the chloroplast and recover quickly from water stress [82]. Similarly, a mutant tobacco plant with a higher photorespiratory capacity (higher glutamine synthase activity) was less susceptible to photoinhibition at 25°C than wild-type plants, whereas a mutant with a lower photorespiratory capacity was more sensitive than the wild-type plants [85].
虽然气孔关闭和低 c 值会降低 CO2 固定率,但它们不会降低光呼吸率。事实上,它在低 c 值时略有增加 [82],因此光呼吸对 NADPH 和 ATP 的需求得维持或增加。因此,光呼吸有助于避免 ETC 的过度减少和在 CO2 固定受低 c 值限制的情况下对光系统的长期损害 [2043]。在气孔关闭的干旱条件下,这可能尤为重要。各种研究表明,光呼吸在干旱期间增加,并提供对光抑制的保护[20,80,82,83,84]。 洋地黄中,水分胁迫使净光合作用减少了 70%;然而,由于光呼吸对 NADPH 和 ATP 的持续需求,以及线粒体甘氨酸脱羧释放的大部分 CO2 被叶绿体中的 Rubisco 重新同化,因此对能量的代谢需求仅减少了 40% [82]。通过维持对这些代谢物的需求,D. lanata 能够避免叶绿体的过度还原,并从水分胁迫中迅速恢复[82]。 同样,在25°C时,具有较高光呼吸能力(较高谷氨酰胺合酶活性)的突变烟草植株比野生型植株更不易受到光抑制,而具有较低光呼吸能力的突变株比野生型植株更敏感[85]。
The fact that mitochondrial activity is essential for the glycine metabolism during photorespiration may be partly why inhibition of the mitochondrial ETC results in increased photoinhibition [32, 70, 71, 72, 73]. In vivo, much of the NADH produced by glycine decarboxylation may be exported to the cytosol via the Mal/OAA shuttle and oxidised in the peroxisome. However, decarboxylation of glycine can contribute to the mitochondrial ETC if the peroxisome requirements for NADH are partly met by glycolysis or the chloroplast. The latter would be likely whenever there was an excess of NADPH in the chloroplast (e.g. when low ci values limit CO2 fixation rates). Krömer and Heldt [86] suggested that only 25–50% of the NADH produced from glycine oxidation in the mitochondria is exported to the peroxisomes. Therefore, 50–75% of the reducing equivalents needed to support the peroxisome requirements for NADH has to come from the chloroplasts.
线粒体活性对于光呼吸过程中甘氨酸代谢至关重要这一事实可能是抑制线粒体 ETC 导致光抑制增加的部分原因 [3270717273].在体内,甘氨酸脱羧产生的大部分 NADH 可以通过 Mal/OAA 穿梭机输出到胞质溶胶中,并在过氧化物酶体中被氧化。然而,如果糖酵解或叶绿体部分满足 NADH 的过氧化物酶体需求,甘氨酸的脱羧可促进线粒体 ETC。每当叶绿体中 NADPH 过量时(例如,当低 c 值限制 CO2 固定率时),就可能出现后者。Krömer 和 Heldt [86] 认为,线粒体中甘氨酸氧化产生的 NADH 中只有 25-50% 输出到过氧化物酶体。因此,支持 NADH 的过氧化物酶体需求所需的 50-75% 的还原当量必须来自叶绿体。
In wheat leaves in vitro NADP+-MDH activity increases following drought treatment [87]. Although this does not necessarily reflect actual changes in the in vivo activity, it does suggest that drought increases use of the Mal/OAA shuttle mechanism to export excess photosynthetic reducing equivalents. These reducing equivalents have to be oxidised elsewhere and in another study on wheat leaves it was found that drought induced an increase in O2 uptake related to the oxidation of photosynthetic reductant [83].
在小麦叶片中,体外 NADP+-MDH 活性在干旱处理后增加 [87]。虽然这不一定反映体内活性的实际变化,但它确实表明干旱增加了 Mal/OAA 穿梭机制的使用,以输出过量的光合还原当量。这些还原当量必须在其他地方被氧化,在另一项关于小麦叶片的研究中发现,干旱诱导了与光合还原剂氧化相关的 O2 吸收增加 [83]。

3.6.3. Mitochondrial activity and protection against photoinhibition
3.6.3. 线粒体活性和对光抑制的保护

There is evidence to suggest that the protective mechanisms against photoinhibition may be different at different temperatures. In the cold, the most important mechanism to prevent photoinhibition appears to be the ability to keep QA relatively oxidised and to avoid damage to the D1 protein of PSII [73]. In addition to the mechanism described in Section 3.5.1, over-reduction of QA can also be avoided via mitochondrial oxidation of excess photosynthetic reducing equivalents [32, 71, 72].
有证据表明,在不同温度下,针对光抑制的保护机制可能不同。在寒冷中,防止光抑制的最重要机制似乎是保持QA相对氧化并避免对PSII的D1蛋白造成损害的能力[73]。除了第3.5.1节中描述的机制外,还可以通过线粒体氧化过量的光合还原当量来避免QA的过度还原[32,71,72]。
At high temperatures and high irradiance, photoinhibition is less dependent on the rate of damage to the D1 protein. Rather, photoinhibition at high temperatures is more dependent on the rate of D1 protein repair [20, 72, 88]. The fact that D1 protein repair is ATP-dependent means that mitochondrial ATP production may contribute to the prevention or minimisation of photoinhibition at high temperatures [70, 71]. The D1 protein is continually repaired and as long as repair can keep up with damage no net photoinhibition will be observed [88]. In cyanobacteria, inhibition of either dark respiration (using azide) or uncoupling of mitochondrial phosphorylation results in an increase in photoinhibition [70], suggesting that prevention of photoinhibition is dependent on mitochondrial ATP synthesis.
在高温和高辐照度下,光抑制对 D1 蛋白损伤速率的依赖性较小。相反,高温下的光抑制更依赖于 D1 蛋白修复的速率 [207288]。D1 蛋白修复是 ATP 依赖性的这一事实意味着线粒体 ATP 的产生可能有助于预防或最小化高温下的光抑制 [7071]。D1 蛋白不断被修复,只要修复能跟上损伤,就不会观察到净光抑制 [88]。在蓝细菌中,抑制暗呼吸(使用叠氮化物)或线粒体磷酸化解偶联导致光抑制增加 [70],表明光抑制的预防取决于线粒体 ATP 合成。

3.6.4. Nutrient limitations and excess photosynthetic reductant
3.6.4. 养分限制和过量的光合还原剂

The imbalance between the production and consumption of NADPH and ATP will be increased under nutrient limiting conditions which may restrict biosynthesis [89, 90]. An excess of NADPH production can therefore occur under conditions of nutrient stress [91, 92]. The fact that the demand for ATP is also low under low nutrient supply may also mean that the processes that oxidise reductant without ATP production might increase in activity (e.g. non-phosphorylating pathways of mitochondrial electron transport) as suggested by several authors [93, 94, 95, 96, 97, 98]. The in vivo involvement of the non-phosphorylating mitochondrial pathways in the light under nutrient limitations or their effect on the redox state of the chloroplast in leaf tissue has not yet been confirmed. On the other hand, an increase in energy dissipation by the xanthophyll cycle under nitrogen limitation has been demonstrated [50].
在可能限制生物合成的营养限制条件下,NADPH 和 ATP 的产生和消耗之间的不平衡将增加 [8990]。因此,在营养胁迫条件下,NADPH 产生过量 [9192]。在低营养供应的情况下,对 ATP 的需求也很低这一事实也可能意味着,正如几位作者所建议的,在没有 ATP 产生的情况下氧化还原剂的过程可能会增加活性(例如线粒体电子传递的非磷酸化途径)[939495969798]。在营养限制下,非磷酸化线粒体途径在光中的体内参与或其对叶组织中叶绿体氧化还原状态的影响尚未得到证实。另一方面,已经证明在氮限制下,叶黄素循环会增加能量耗散[50]。

3.7. Role of mitochondria in providing carbon skeletons in the light
3.7. 线粒体在光下提供碳骨架中的作用

In addition to producing ATP and oxidising excess photosynthetic reducing equivalents, mitochondria serve another important role in the light: production of carbon intermediates for biosynthesis (e.g. the production of 2-OG and/or citrate). Most researchers prior to the 1990s assumed that mitochondria exported 2-OG. However, more recent work suggests that citrate is the primary carbon skeleton exported [10]. For example, when spinach leaf mitochondria are incubated in a medium with a composition similar to the cytosol in the light, the main product of malate oxidation is citrate [10]. Citrate can be converted in the cytosol to 2-OG [6, 99] (see 2.3 Exchange of carbon compounds, Fig. 3) as the precursor for glutamate and glutamine formation [10].
除了产生 ATP 和氧化过量的光合还原当量外,线粒体在光中还起着另一个重要作用:生产用于生物合成的碳中间体(例如生产 2-OG 和/或柠檬酸盐)。1990 年代之前的大多数研究人员认为线粒体输出 2-OG。然而,最近的研究表明,柠檬酸盐是出口的主要碳骨架 [10]。例如,当菠菜叶线粒体在光照下成分类似于胞质溶胶的培养基中孵育时,苹果酸氧化的主要产物是柠檬酸盐[10]。柠檬酸盐可以在胞质溶胶中转化为 2-OG [699](参见 2.3 碳化合物的交换图 3),作为谷氨酸和谷氨酰胺形成的前体 [10]。
Many processes require carbon skeletons from the mitochondria, of which nitrogen assimilation is the most important [61, 100]. The different pathways by which carbon can enter and leave the mitochondria enable the mitochondria to be flexible in their supply of carbon skeletons.
许多过程需要来自线粒体的碳骨架,其中氮同化是最重要的[61,100]。 碳可以进入和离开线粒体的不同途径使线粒体能够灵活地供应碳骨架。

3.8. Rates of O2 uptake and CO2 release in light versus darkness
3.8. 光明与黑暗中 O2 摄取和 CO2 释放的速率

In darkness, there are several phases to respiration including glycolysis, the TCA cycle and the oxidation of NADH and FADH2. Gas exchange occurs in two of these processes: CO2 release from decarboxylation reactions in the TCA cycle and O2 uptake related to oxidation of NAD(P)H and FADH2 in the mitochondrial ETC. Measurements of respiration (O2 uptake or CO2 release) in photosynthetic tissues in the dark are relatively straight forward, with the ratio of O2 uptake to CO2 release (respiratory quotient, RQ) typically being between 0.8 and 1.6 ([101] and refs. therein).
在黑暗中,呼吸有几个阶段,包括糖酵解、TCA 循环以及 NADH 和 FADH2 的氧化。气体交换发生在其中两个过程中:TCA 循环中脱羧反应中 CO2 的释放和与线粒体中 NAD(P)H 和 FADH2 氧化相关的 O2 摄取 ET。在黑暗中光合组织中呼吸(O2 摄取或 CO2 释放)的测量相对简单, O2 摄取与 CO2 释放(呼吸商,RQ)的比率通常在 0.8 和 1.6 之间([101] 和参考文献)。
Measurements of respiratory gas exchange in the light are not so straightforward, because photosynthetic, photorespiratory and respiratory processes occur at the same time. Photorespiratory and non-photorespiratory reactions result in mitochondrial O2 consumption, while O2 is produced by photosynthesis. O2 is also consumed in the chloroplast as a result of photorespiration and the Mehler reactions [102]. Photosynthesis and PEP carboxylase result in CO2 uptake at the same time that CO2 is released in the mitochondria by photorespiration and the TCA cycle, in addition to CO2 released by the oxidative pentose phosphate pathway. If the TCA cycle is differently affected by light than is mitochondrial electron transport, the effect of light on CO2 release will differ from that on O2 uptake. For example, oxidation of excess photosynthetic reducing equivalents by the mitochondria may be coupled to O2 uptake but not to CO2 release.
在光中测量呼吸气体交换并不那么简单,因为光合作用、光呼吸和呼吸过程同时发生。光呼吸和非光呼吸反应导致线粒体 O2 消耗,而 O2 由光合作用产生。由于光呼吸和梅勒反应,O2 也在叶绿体中被消耗 [102]。光合作用和 PEP 羧化酶导致 CO2 吸收,同时 CO2 通过光呼吸和 TCA 循环在线粒体中释放,此外,CO2 由氧化磷酸戊糖途径释放。如果 TCA 循环受光的影响与线粒体电子传递的影响不同,则光对 CO2 释放的影响将与对 O2 摄取的影响不同。例如,线粒体对过量光合还原当量的氧化可能与 O2 摄取耦合,但与 CO2 释放无关。
Despite the problems in determining respiratory gas exchange in the light, numerous studies have used gas exchange and mass spectrometry techniques to measure respiration in the light. In all studies, respiration continued in the light. However, the degree to which it continued depended strongly on whether CO2 release or O2 uptake was measured. Variations in experimental conditions and plant species also contribute to the variability in the estimates of respiration in the light.
尽管在确定光线下的呼吸气体交换存在问题,但许多研究已经使用气体交换和质谱技术来测量光线下的呼吸。在所有研究中,呼吸在光线下继续进行。然而,它持续的程度在很大程度上取决于是否测量了 CO2 释放或 O2 吸收。实验条件和植物种类的变化也会导致光下呼吸估计的可变性。
The effects of light on mitochondrial O2 uptake are not uniform, varying from partial inhibition [103, 104], no change [31, 105] to a substantial increase [106]. The variability in mitochondrial O2 consumption in the light may reflect variability in the supply of substrate to the mitochondria (e.g. glycolytic products and excess photosynthetic reducing equivalents) and the degree to which photorespiratory NADH is oxidised in the mitochondria (see Section 3.6.2). It may also reflect variability in the demand for respiratory ATP by cellular processes in the light.
光对线粒体 O2 摄取的影响并不均匀,从部分抑制 [103104]、无变化 [31105] 到大幅增加 [106]。光中线粒体 O2 消耗的可变性可能反映了线粒体底物供应的可变性(例如 糖酵解产物和过量的光合还原当量)以及光呼吸 NADH 在线粒体中被氧化的程度(参见第 3.6.2 节)。它还可能反映细胞过程在光线下对呼吸 ATP 需求的变化。
The effect of light on CO2 release is more clear. Under photorespiratory conditions, total mitochondrial CO2 release is higher in the light than in darkness due to the combined release of CO2 by glycine decarboxylation and non-photorespiratory processes (e.g. TCA cycle [17]). However, non-photorespiratory CO2 release is lower in the light than in darkness in most species investigated, with the degree of inhibition by light ranging from 25 to 75% in studies using mass spectrometry [31, 72, 107, 108] and gas exchange techniques [108, 109, 110, 111, 112, 113, 114].
光对 CO2 释放的影响更加明显。在光呼吸条件下,由于甘氨酸脱羧和非光呼吸过程(例如 TCA 循环 [17])共同释放 CO2,线粒体总 CO2 在光明下比在黑暗中更高。然而,在所研究的大多数物种中,非光呼吸 CO2 在光下的释放低于在黑暗中,在使用质谱 [3172107108] 和气体交换技术 [108109110] 的研究中,光的抑制程度为 25% 至 75%111112113114]。

3.9. Mechanisms responsible for inhibition of CO2 release in the light
3.9. 抑制 CO2 在光线下释放的机制

The mechanism responsible for light inhibition of non-photorespiratory CO2 release is unresolved. However, Atkin et al. [114] recently suggested that light inhibition of respiration may be the result of the inactivation of PDC and NAD+-ME in the light [115, 116, 117, 118, 119]. PDC and NAD+-ME determine the flux of carbon into the TCA cycle [119] (Fig. 3). While the mechanism responsible for the light inhibition of NAD+-ME is not known, the inhibition of PDC is clearly the result of phosphorylation [115, 116]. The inhibition of PDC activity mainly occurs under photorespiratory conditions [117, 120]. The photorespiration-dependent inhibition of PDC may be enhanced by NH3 (produced during glycine decarboxylation) stimulating the protein kinase that phosphorylates PDC [6, 115]. Increased ATP synthesis due to increased electron transport during glycine oxidation could also contribute to PDC inactivation [118].
负责光抑制非光呼吸 CO2 释放的机制尚未解决。然而,Atkin等[114]最近提出,光对呼吸的抑制可能是PDC和NAD+-ME在光下失活的结果[115,116,117,118,119]。 PDC 和 NAD+-ME 决定了碳进入 TCA 循环的通量 [119](图 3)。虽然负责光抑制 NAD+-ME 的机制尚不清楚,但 PDC 的抑制显然是磷酸化的结果 [115116]。PDC 活性的抑制主要发生在光呼吸条件下 [117120]。NH3(在甘氨酸脱羧过程中产生)刺激磷酸化 PDC 的蛋白激酶,可能会增强 PDC 的光呼吸依赖性抑制 [6115]。甘氨酸氧化过程中电子传递增加导致 ATP 合成增加也可能导致 PDC 失活 [118]。
The apparent light inhibition of non-photorespiratory CO2 release may also be partly the result of reduced flux through glycolysis in the light. For example, pyruvate kinase and PEPC activities are lower in the light than in darkness in the green alga Chlamydomonas [106]. PDC activity was 25% lower in the light than in darkness in this species.
非光呼吸 CO2 释放的明显光抑制也可能部分是由于在光中通过糖酵解减少通量的结果。例如,绿藻衣藻中的丙酮酸激酶和 PEPC 活性在光照下低于在黑暗中 [106]。该物种在光照下的 PDC 活性比在黑暗中低 25%。
Another factor that may be partly responsible for light inhibition of non-photorespiratory CO2 release are enhanced rates of export of TCA cycle carbon intermediates to the cytosol to support light-dependent nitrogen assimilation [121]. TCA cycle CO2 release could be reduced under conditions where 2-OG and/or citrate are exported from the mitochondrion to support amino acid synthesis. Removal of these would eliminate one site of TCA cycle CO2 release. This hypothesis, which remains to be tested, is supported by the fact that the CO2 compensation point of barley leaves increases when plants are transferred from NO3 to NH+4 nutrient [122]. NH+4 is not transported from roots to shoots but rather is assimilated in the roots. This eliminates leaf nitrogen assimilation, thereby decreasing the demand for TCA cycle intermediates in the leaves (and increase the rate of CO2 release and CO2 compensation point).
另一个可能部分导致光抑制非光呼吸 CO2 释放的因素是 TCA 循环碳中间体向胞质溶胶输出的速率增加,以支持光依赖性氮同化 [121]。在从线粒体输出 2-OG 和/或柠檬酸盐以支持氨基酸合成的条件下,可以减少 TCA 循环 CO2 的释放。去除这些将消除 TCA 循环 CO2 释放的一个位点。这一假设仍有待检验,但当植物从NO-3营养物质转移到NH+4营养物质时,大麦叶片的CO2补偿点会增加[122]这一事实也支持了这一假设。NH+4 不是从根运输到芽,而是在根中被同化。这消除了叶片氮的同化,从而减少了叶片中对 TCA 循环中间体的需求(并增加了 CO2 释放速率和 CO2 补偿点)。

3.10. Effect of light-to-dark transitions on respiration
3.10. 明暗转变对呼吸的影响

The fact that non-photorespiratory CO2 release is lower in the light than in darkness suggests that light-to-dark transitions might result in a direct increase in CO2 release until steady state dark respiration values are achieved. This is, however, rarely the case. When first exposed to darkness following a period in the light, leaves often exhibit transient increases in dark respiration before steady state values are achieved. The first transient increase (after approx. 15–20 s of darkness [114]) is the photorespiratory post-illumination burst (PIB), while the second (180–250 s [114]) has been defined as light enhanced dark respiration (LEDR [123]).
非光呼吸 CO2 在光中释放量低于在黑暗中,这一事实表明,从明到暗的转变可能导致 CO2 释放量直接增加,直到达到稳态暗呼吸值。然而,这种情况很少见。当在光照下一段时间后首次暴露在黑暗中时,叶子通常会在达到稳态值之前表现出黑暗呼吸的瞬态增加。第一次瞬变增加(在大约15-20 s的黑暗后[114])是光呼吸照明后突发(PIB),而第二次(180-250 s [114])被定义为光增强暗呼吸(LEDR [123])。
PIB occurs because of a difference in time that the RuBP and glycine pools remain in the cell following the transition to darkness. CO2 fixation by Rubisco consumes the RuBP within 30 s [122] while the glycine pool initially remains stable (for 15–20 s) before declining. The continued decarboxylation of glycine is observed as a burst of CO2 release.
PIB 的发生是因为 RuBP 和甘氨酸池在过渡到黑暗后留在细胞中的时间不同。Rubisco 固定的 CO2 在 30 s 内消耗 RuBP [122],而甘氨酸池最初保持稳定(15-20 s),然后下降。甘氨酸的持续脱羧表现为 CO2 释放的爆发。
LEDR has been reported as increased O2 consumption [106, 119, 123, 124, 125, 126] and CO2 evolution [78, 112, 127]. It takes about 3–5 min for LEDR to reach its maximum rate in darkness. It appears to reflect the initially high concentration of photosynthetic metabolites immediately available to the mitochondria (e.g. pyruvate or malate) in darkness after a period of illumination [72]. LEDR also appears to be associated with reversal of light inhibition of key enzymes (e.g. pyruvate dehydrogenase complex, PDC and NAD+-ME) that control entry of carbon into the mitochondrial TCA cycle [119]. The magnitude of LEDR is dependent on the size of the substrate pool at the end of the light period. This pool size reflects two things: firstly, the rate and duration of photosynthesis in the preceding period and, secondly, the rate of substrate consumption (e.g. by respiration) during the light period, which will be affected by the degree of light inhibition of the key enzymes of pathways that use photosynthetic products (e.g. PDC and NAD-ME). This hypothesis is supported by recent work that shows that the degree of inhibition of leaf respiration by light closely matches the magnitude of LEDR, and that LEDR and light inhibition of leaf respiration are equally sensitive to increasing irradiances in the light period [114]. Moreover, both parameters are insensitive to light quality and are tightly correlated [106, 114].
据报道,LEDR 表现为 O2 消耗量增加 [106119123124125126] 和 CO2 释放 [78112127].LEDR 在黑暗中达到最大速率大约需要 3-5 分钟。它似乎反映了在光照一段时间后,线粒体在黑暗中立即可用的光合代谢物(例如丙酮酸或苹果酸)的初始高浓度[72]。LEDR 似乎还与控制碳进入线粒体 TCA 循环的关键酶(例如丙酮酸脱氢酶复合物、PDC 和 NAD+-ME)的光抑制逆转有关[119]。LEDR 的幅度取决于光照周期结束时衬底池的大小。这个池大小反映了两件事:首先,前一时期的光合作用速率和持续时间,其次,光照期间底物消耗的速率(例如通过呼吸),这将受到使用光合作用产物的途径的关键酶(例如 PDC 和 NAD-ME)的光抑制程度的影响。这一假设得到了最近的研究的支持,该研究表明,光对叶片呼吸的抑制程度与LEDR的大小非常匹配,并且LEDR和叶片呼吸的光抑制对光照周期中增加的辐照度同样敏感[114]。 此外,这两个参数对光质量不敏感,并且密切相关[106,114]。

4. Interactions between chloroplasts and mitochondria in the dark
4. 叶绿体与线粒体在黑暗中的相互作用

Interactions between mitochondria and chloroplasts in photosynthetic cells also occur in the dark, as demonstrated by the fact that inhibition of mitochondrial activity in the dark affects the PQ redox state and the thylakoid electrochemical gradient [128, 129]. In the dark, mitochondria are the main source of ATP for cell processes, including those in the chloroplasts, which, although not photosynthesising, are still metabolically active, e.g. starch that has been accumulated in the light needs to be converted to hexose-P and TP and exported to the cytosol [130]. In the dark, mitochondrial ATP, and sometimes reductant, might also be necessary to prepare the chloroplast for optimal photosynthetic activity when the light returns, by maintaining a proton gradient across the thylakoid membrane or by poising the PQ pool [131].
光合细胞中线粒体和叶绿体之间的相互作用也发生在黑暗中,黑暗中线粒体活性的抑制会影响PQ氧化还原状态和类囊体电化学梯度[128,129]。 在黑暗中,线粒体是细胞过程中ATP的主要来源,包括叶绿体中的ATP,叶绿体虽然没有进行光合作用,但仍然具有代谢活性,例如,在光照下积累的淀粉需要转化为己糖-P和TP并输出到胞质溶胶中[130]。在黑暗中,线粒体ATP(有时是还原剂)也可能是必需的,以通过维持类囊体膜上的质子梯度或通过使PQ池保持稳定[131],使叶绿体在光线返回时为最佳光合作用活性做好准备[131]。

4.1. Mitochondrial ATP maintains the thylakoid proton gradient
4.1. 线粒体 ATP 维持类囊体质子梯度

In Chlorella in the dark the electrochemical gradient across the thylakoid membrane can be sustained or restored by ATP (supplied by the mitochondria) through reverse operation of the ATPase [131] (Fig. 4). Similarly, in higher plants ATP hydrolysis can maintain a proton gradient across the thylakoid membrane in the dark [132]. Although chloroplast proton gradients are not maintained in the dark under favourable growth conditions, they are maintained in the dark following exposure to photoinhibitory cold, bright conditions [132]. This maintenance of a dark proton gradient may be important to allow non-radiative dissipation by the xanthophyll cycle, offering photoprotection by non-radiative dissipation upon re-illumination. The degree to which the ATPase remains active in the dark is dependent on the levels of zeaxanthin and violaxanthin in the lumen and the temperature [132]. At high temperatures the ATPase is inactivated within minutes in the dark to avoid wasteful ATP hydrolysis [132]. In contrast, the ATPase can remain active for hours in the dark at low temperatures, even overnight [132]. Although the ATP requirement for maintaining the proton gradient is low, maintenance of ATPase activity may partly explain why cold hardening of plants results in higher respiration rates at a given temperature [32].
在黑暗中的小球藻中,通过 ATP 酶的反向操作,ATP(由线粒体提供)可以维持或恢复跨类囊体膜的电化学梯度 [131](图 4)。同样,在高等植物中,ATP 水解可以在黑暗中保持质子梯度穿过类囊体膜 [132]。虽然在有利的生长条件下,叶绿体质子梯度在黑暗中不维持,但在暴露于光抑制性寒冷、明亮的条件下后,它们在黑暗中保持不变[132]。这种暗质子梯度的维持对于允许叶黄素循环的非辐射耗散可能很重要,从而在重新照明时通过非辐射耗散提供光保护。ATP酶在黑暗中保持活性的程度取决于管腔中玉米黄质和紫黄质的水平以及温度[132]。在高温下,ATP酶在黑暗中在几分钟内失活,以避免浪费的ATP水解[132]。相比之下,ATP酶可以在低温下在黑暗中保持活性数小时,甚至过夜[132]。尽管维持质子梯度的 ATP 要求很低,但维持 ATP 酶活性可能部分解释了为什么植物的冷硬化在给定温度下会导致更高的呼吸速率 [32]。

4.2. Reduction of PQ by NAD(P)H
4.2. NAD(P)H 降低 PQ

For chloroplasts to function in the light, it is important that PQ remains partly reduced in the dark to provide electrons for PSI upon re-illumination [133]. The reducing equivalents needed for this are supplied by the mitochondria and/or by starch degradation in the chloroplast [46, 134]. Reduction of the PQ pool in the dark has been reported for both higher plants and algae [135, 136, 137, 138, 139] and active re-reduction of PQ is observed in the dark after oxidation by far red light [137, 140]. Reduction of PQ by NAD(P)H may be mediated by a NAD(P)H-PQ oxidoreductase located in the thylakoid membrane [128]. Several lines of evidence indicate the existence of an NAD(P)H-PQ oxidoreductase in the chloroplast of both green algae [129, 141, 142, 143] and higher plants [140, 144]. In addition, 11 open reading frames showing great similarity with parts of complex I (a mitochondrial NADH-Q oxidoreductase) have been found in the chloroplast genome [134, 145]. Isolated thylakoid membranes have also been shown to oxidise NAD(P)H in the presence of several electron acceptors, such as ferricyanide and benzoquinone [134]. Although an enzyme with demonstrated NADPH-PQ activity has not been purified or isolated thus far, a large protein complex with NAD(P)H to nitrotetrazolium blue oxidoreductase activity was isolated from barley thylakoids [146]. Also the reduction of PQ has been shown to be inhibited by rotenone, an inhibitor of complex I [142, 147].
为了使叶绿体在光照下发挥作用,重要的是PQ在黑暗中保持部分还原,以便在重新照明时为PSI提供电子[133]。为此所需的还原当量由线粒体和/或叶绿体中的淀粉降解提供[46,134]。 据报道,高等植物和藻类在黑暗中 PQ 池减少 [135136137138139],并且在远红光氧化后在黑暗中观察到 PQ 的主动再还原 [137140].NAD(P)H降低PQ可能是由位于类囊体膜中的NAD(P)H-PQ氧化还原酶介导的[128]。几条证据表明,绿藻 [129141142143] 和高等植物 [140144] 的叶绿体中存在 NAD(P)H-PQ 氧化还原酶].此外,在叶绿体基因组中发现了 11 个开放阅读框,与复合物 I(一种线粒体 NADH-Q 氧化还原酶)的部分内容非常相似 [134145]。分离的类囊体膜也被证明在几种电子受体(如铁氰化物和苯醌)存在下氧化 NAD(P)H [134]。 虽然到目前为止尚未纯化或分离出具有 NADPH-PQ 活性的酶,但从大麦类囊体中分离出一种具有 NAD(P)H 与硝基四唑蓝氧化还原酶活性的大蛋白复合物 [146]。此外,鱼藤酮(复合物 I 的抑制剂)也显示 PQ 的降低受到抑制 [142147]。

4.3. Interaction between mitochondrial activity and PQ redox state
4.3. 线粒体活性与 PQ 氧化还原状态之间的相互作用

The redox level of the chloroplast PQ pool in the dark responds strongly to mitochondrial activity. Inhibition of mitochondrial phosphorylation (via uncoupling, anaerobiosis or by inhibition of mitochondrial electron transport or ATPase activity) in the dark often results in an increase in the oxidation state of the chloroplast PQ pool [128, 136, 148]. For Chlamydomonas strong evidence was presented to suggest that the reduction of PQ was mediated by an increase in glycolysis [128, 148]. Inhibition of mitochondrial phosphorylation lowers cellular ATP levels, resulting in an increase in glycolytic activity (via the Pasteur effect), leading to increased NADPH production in the chloroplast. In Chlamydomonas, oxidation of hexose-P to 3-PGA (the initial stage of glycolysis) occurs in the chloroplast [149]. In higher plants glycolysis occurs in the cytosol and redox equivalents are transported into the chloroplast by the Mal/OAA or DHAP/3PGA shuttles. However, the reduction of PQ in higher plants is likely to occur in a manner similar to that in Chlamydomonas, especially since PQ reduction also responds to lowering of intracellular ATP. PQ reduction in tobacco protoplasts was stimulated when respiration was inhibited by KCN, probably increasing the rate of glycolysis by the Pasteur effect [140]. In spinach leaves it was found that PQ reduction in the dark was dependent on a reductant from the cytosol [136].
叶绿体 PQ 池在黑暗中的氧化还原水平对线粒体活性有强烈反应。在黑暗中抑制线粒体磷酸化(通过解偶联、厌氧或抑制线粒体电子传递或 ATP 酶活性)通常会导致叶绿体 PQ 库的氧化态增加 [128136148]。对于衣藻,有力的证据表明 PQ 的降低是由糖酵解的增加介导的 [128148]。抑制线粒体磷酸化降低细胞 ATP 水平,导致糖酵解活性增加(通过巴斯德效应),导致叶绿体中 NADPH 产生增加。在衣藻中,己糖-P 氧化成 3-PGA(糖酵解的初始阶段)发生在叶绿体中 [149]。在高等植物中,糖酵解发生在胞质溶胶中,氧化还原当量通过 Mal/OAA 或 DHAP/3PGA 穿梭机运输到叶绿体中。然而,高等植物中 PQ 的降低可能以类似于衣藻的方式发生,特别是因为 PQ 降低也对细胞内 ATP 的降低做出反应。当 KCN 抑制呼吸时,会刺激烟草原生质体中 PQ 的减少,这可能通过巴斯德效应增加糖酵解速率 [140]。在菠菜叶中,发现黑暗中PQ的降低取决于胞质溶胶的还原剂[136]。
The movement of LHCs from one photosystem to the other (i.e. state transitions; Section 3.5.2) also occur in the dark [46] and respond to mitochondrial activity. For example, lowering of the chloroplast ATP concentration by inhibiting ATP mitochondrial synthesis in the dark (by uncoupling or inhibiting respiration) can result in a transition from state I to state II [46]. A decrease in the level of ATP in the cell is usually accompanied by a reduction of the ETC between the photosystems. The transition from state I to state II is regulated by the redox state of PQ [45] and is probably a consequence of the reduction of PQ. For a return to state I both oxidation of the ETC and a high ATP level are essential [46]. The state transition is suggested to prepare the chloroplast for cyclic phosphorylation [46] or to prevent over-reduction of the ETC between the photosystems when the light returns [150].
LHC 从一个光系统到另一个光系统的运动(即状态转换;节 3.5.2)也发生在黑暗中[46],并对线粒体活动有反应。例如,通过在黑暗中抑制 ATP 线粒体合成(通过解耦或抑制呼吸)来降低叶绿体 ATP 浓度,可导致从状态 I 过渡到状态 II [46]。细胞中 ATP 水平的降低通常伴随着光系统之间 ETC 的减少。从状态 I 到状态 II 的转变受 PQ 的氧化还原状态调节 [45],可能是 PQ 降低的结果。为了恢复到状态 I,ETC 的氧化和高 ATP 水平都是必不可少的 [46]。建议进行状态转换以准备叶绿体进行循环磷酸化[46],或防止光返回时光系统之间的ETC过度减少[150]。
Taken together, the above studies demonstrate the strong interaction between mitochondria and chloroplasts in the dark.
综上所述,上述研究表明,线粒体和叶绿体在黑暗中具有很强的相互作用。

4.4. Chlororespiration 4.4. 氯呼吸

4.4.1. Overall characteristics of chlororespiration
4.4.1. 氯呼吸的总体特征

The reduction PQ in the dark may represent the first step of chlororespiration (CR [141]). The term chlororespiration was introduced for a proposed electron transport pathway consuming O2 in the thylakoid membrane. CR is thought to represent the oxidation of NAD(P)H, involving an NAD(P)H-PQ oxido-reductase and a PQ oxidase (Fig. 4) and could explain the reduction of PQ in the dark and its increased reduction upon anaerobiosis [141]. Although considerable evidence for an NAD(P)H-PQ oxido-reductase has been found [129, 140, 141, 142, 143], evidence for a PQ oxidase is lacking [129, 134, 147]. In Chlamydomonas it was shown that O2 uptake was related to reduction of the PQ pool [151, 152]. Most evidence for CR has been found in Chlamydomonas, although the existence of CR in higher plants has also been suggested [135, 140, 147, 153, 154]. However, the evidence for CR in higher plant chloroplasts is limited to reduction of PQ in the dark, rather than O2 uptake in association with CR. The existence of a PQ oxidase in higher plants has been suggested [147] but based only on PQ reduction data and using inhibitors, which can lead to ambiguous results (see Section 4.4.2).
黑暗中PQ的降低可能代表氯呼吸作用的第一步(CR [141])。术语氯呼吸是指在类囊体膜中消耗 O2 的拟议电子传递途径。CR被认为代表NAD(P)H的氧化,涉及NAD(P)H-PQ氧化还原酶和PQ氧化酶(图4),可以解释PQ在黑暗中的减少和厌氧后减少的增加[141]。尽管已经发现了 NAD(P)H-PQ 氧化还原酶的大量证据 [129140141142143],但缺乏 PQ 氧化酶的证据 [129134147]。在衣藻中,研究表明 O2 摄取与 PQ 库的减少有关 [151152]。大多数CR的证据是在衣藻中发现的,尽管也有人认为CR存在于高等植物中[135,140,147,153,154]。 然而,高等植物叶绿体中 CR 的证据仅限于在黑暗中降低 PQ,而不是与 CR 相关的 O2 摄取。 有人提出PQ氧化酶存在于高等植物中[147],但仅基于PQ还原数据和使用抑制剂,这可能导致模棱两可的结果(参见第4.4.2节)。
Other components of the chloroplast ETC, such as cytochrome b6f complex and plastocyanin (PC, Fig. 4) are not believed to be involved in chlororespiration, because CR is not sensitive to DBMIB (2-nonyl-4-hydroxyquinoline N-oxide) an inhibitor of electron transport between PQ and cytochrome b6f complex. Moreover, CR still occurs in mutants of Chlamydomonas deficient in cytochrome b6f complex or photosystem I [151]. On the other hand, electrons flowing from PSII to PQ can be used in CR as shown by the fact that DCMU (3-(3,4-dichlorophenyl)-1,1-dimethyl urea) inhibits the PSII dependent O2 uptake in a mutant deficient in PSI [151]. So, the only components involved in CR appear to be a NAD(P)H dehydrogenase (or NAD(P)H-PQ reductase), PQ and a putative PQ-oxidase [129] (Fig. 4).
叶绿体 ETC 的其他成分,如细胞色素 b6f 复合物和质体蓝蛋白(PC,图 4)被认为不参与氯呼吸作用,因为 CR 对 DBMIB(2-壬基-4-羟基喹啉 N-氧化物)不敏感,DBMIB 是 PQ 和细胞色素 b6f 复合物之间的电子传递抑制剂。此外,CR仍然发生在缺乏细胞色素b6f复合物或光系统I的衣藻突变体中[151]。另一方面,从PSII流向PQ的电子可用于CR,DCMU(3-(3,4-二氯苯基)-1,1-二甲基脲)抑制PSI缺乏的突变体中PSII依赖性O2的摄取[151]。因此,CR 中唯一涉及的成分似乎是 NAD(P)H 脱氢酶(或 NAD(P)H-PQ 还原酶)、PQ 和推定的 PQ 氧化酶 [129](图 4)。
Apart from the NAD(P)H-dehydrogenase and the PQ oxidase, it has been suggested that CR activity depends on a proton gradient across the thylakoid membrane [129, 141, 155]. This has been used to explain the inhibition of CR by the ionophore, dicyclohexyl-18-crown-6, an uncoupler of photophosphorylation [141, 155].
除了 NAD(P)H-脱氢酶和 PQ 氧化酶外,有人认为 CR 活性取决于类囊体膜上的质子梯度 [129141155]。这已被用来解释离子载体二环己基-18-冠-6(光磷酸化的解偶联剂)对 CR 的抑制 [141155]。

4.4.2. Inhibitors of chlororespiration
4.4.2. 氯呼吸抑制剂

Experimental testing of the model of chlororespiration is not straightforward. One problem is that chlororespiration does not have an unique feature that can be measured, e.g. it shares O2 uptake with Mehler reactions, mitochondrial respiration and photorespiration. Furthermore, a change in the PQ redox state does not necessarily reflect changes in chlororespiration activity. Also, mitochondrial and chlororespiratory enzymes are often sensitive to the same class of inhibitors and their use can lead to ambiguous results [129]. For example, although myxothiazol was thought to inhibit CR [156], CR was found to be insensitive to this inhibitor in a mutant of Chlamydomonas in which the mitochondrial cytochrome bc1 complex was resistant to myxothiazol [129].
氯呼吸模型的实验测试并不简单。一个问题是氯呼吸没有可以测量的独特特征,例如它与 Mehler 反应、线粒体呼吸和光呼吸共享 O2 摄取。此外,PQ 氧化还原状态的变化并不一定反映氯呼吸活性的变化。此外,线粒体酶和叶咽酶通常对同一类抑制剂敏感,使用它们会导致结果模棱两可[129]。例如,尽管粘噻唑被认为抑制 CR [156],但在衣突变体中发现 CR 对这种抑制剂不敏感,其中线粒体细胞色素 bc1 复合物对粘噻唑耐药 [129]。
In addition to myxothiazol, various inhibitors of mitochondrial respiration such as antimycin A [156], KCN [141, 157], CO [141, 147] and SHAM [141] have been suggested to inhibit CR. If correct, KCN would be expected to inhibit CR in all chloroplasts. However, while KCN inhibits CR in Chlamydomonas, no such inhibition is seen in Chlorella [141]. KCN (and other inhibitors of cytochrome oxidase) itself can induce CR via increases in the redox state of PQ [128, 136, 148]. The efficacy of KCN as a CR inhibitor, therefore, remains in doubt. Similar doubts exist for the other inhibitors and conclusions based on their effects must be considered with care. At the moment there is no single compound which has been shown to be an undisputed inhibitor of CR.
除粘液噻唑外,各种线粒体呼吸抑制剂,如抗霉素 A [156]、KCN [141157]、CO [141147] 和 SHAM [141] 也被建议抑制 CR。如果正确,预计 KCN 将抑制所有叶绿体中的 CR。然而,虽然 KCN 抑制衣中的 CR,但在小球藻中未观察到这种抑制 [141]。KCN(和其他细胞色素氧化酶抑制剂)本身可以通过增加PQ的氧化还原状态来诱导CR[128,136,148]。 因此,KCN 作为 CR 抑制剂的疗效仍存疑。对于其他抑制剂也存在类似的疑问,必须谨慎考虑基于其效果的结论。目前,没有单一化合物已被证明是无可争议的 CR 抑制剂。

4.4.3. Role of chlororespiration and in vivo activity
4.4.3. 氯呼吸的作用和体内活性

With the model of CR still unconfirmed one can only speculate on the role of chlororespiration. CR has been suggested to be an adaptation to N-limitation in Chlamydomonas, because CR-dependent O2 consumption increased under N-limitation, concomitantly NADPH-PQ oxido-reductase increased 7-fold [158]. Chlororespiration can facilitate NADPH oxidation to dissipate photosynthetic reducing equivalents and thus minimise photoinhibition or prevent the production of active oxygen species [158]. Such a role would be comparable to that suggested for the mitochondrial alternative oxidase [159]. Another role that has been suggested is the recycling of NADP+ for starch degradation [129, 147].
由于 CR 的模型仍未得到证实,我们只能推测氯呼吸的作用。CR被认为是衣对N限制的适应,因为CR依赖性O2消耗在N限制下增加,同时NADPH-PQ氧化还原酶增加7倍[158]。氯呼吸作用可以促进 NADPH 氧化以消散光合还原当量,从而最大限度地减少光抑制或阻止活性氧的产生 [158]。这种作用与线粒体替代氧化酶的作用相当[159]。另一个被提出的作用是回收 NADP+ 用于淀粉降解 [129147]。
The in vivo activity of CR is also unclear. Maximum activity of CR (i.e. when PQ is completely reduced after inhibition of mitochondrial respiration) is 10–20% of total respiration [141, 157]. It is possible that the small O2 uptake by CR is the result of non-enzymatic oxidation of PQ without any in vivo significance. For experimental data to be conclusive about the activity of a PQ oxidase measurements will need to include rates of O2 uptake, because PQ reduction levels can be affected by many factors. It seems essential that the components, and especially the oxidase, are isolated and characterised, before the model of CR can be accepted.
CR 的体内活性也不清楚。CR 的最大活性(即当 PQ 在抑制线粒体呼吸后完全降低时)为总呼吸的 10-20% [141157]。CR 对 O2 的小摄取可能是 PQ 非酶促氧化的结果,没有任何体内意义。要使实验数据对 PQ 的活性做出结论,氧化酶测量需要包括 O2 摄取速率,因为 PQ 降低水平会受到许多因素的影响。在接受 CR 模型之前,分离和表征成分,尤其是氧化酶,似乎是必不可少的。

5. Concluding remarks 5. 结语

The above discussion demonstrates the interdependence of chloroplasts and mitochondria and the importance of respiration to photosynthesis. The role of mitochondria in the light can vary strongly depending on the conditions. Mitochondrial ATP production may be important for maximum photosynthesis, but an important question is whether this occurs only under conditions favourable for biosynthesis or is more general.
上述讨论证明了叶绿体和线粒体的相互依赖性以及呼吸对光合作用的重要性。线粒体在光线下的作用可能因条件而异。线粒体 ATP 的产生对于最大光合作用可能很重要,但一个重要的问题是这是否仅在有利于生物合成的条件下发生,还是更普遍。
Under adverse conditions, such as drought, high light and/or low temperatures, mitochondria may allow the photosynthetic activity to continue without a net gain of carbon or energy for the cell. This would help a plant to avoid photoinhibition and structural damage (e.g. chlorophyll bleaching) to the photosynthetic apparatus via dissipation of light energy. High leaf respiration rates may thus be a feature of plants exposed to adverse conditions. Indeed inherently slower growing species, characteristic of harsh environments, exhibit relatively high respiration rates compared with fast-growing species characteristic of favourable sites [78]. Cold hardening of plants also increases respiratory capacity [32]. The importance of respiration under stress conditions has thus far only been based on circumstantial (albeit strong) evidence and future research should be directed to obtain more direct evidence. Especially the role of the non-phosphorylating pathways, under those conditions, needs to be established.
在不利条件下,例如干旱、高光和/或低温,线粒体可以允许光合作用活动继续进行,而细胞没有净碳或能量增加。这将有助于植物避免光能耗散对光合装置的光抑制和结构损伤(例如叶绿素漂白)。因此,高叶片呼吸速率可能是暴露于不利条件下的植物的一个特征。事实上,与有利地点的快速生长物种特征相比,恶劣环境特征的天生生长较慢的物种表现出相对较高的呼吸速率[78]。植物的冷硬化也会增加呼吸能力[32]。到目前为止,在压力条件下呼吸的重要性仅基于间接(尽管很强)证据,未来的研究应指向获得更直接的证据。特别是需要确定非磷酸化途径在这些条件下的作用。
If citrate and not 2-OG is the main organic acid exported by the mitochondria (see Section 3.7), extra reducing equivalents (which may be needed for nitrate reduction) are produced in the cytosol. This would change our understanding of mitochondrial metabolism and emphasise the importance of cytosolic NADP+-dependent isocitrate dehydrogenase. Further evidence, possibly involving transgenic plants, is required to establish this; e.g. transgenic plants without cytosolic NADP+-dependent isocitrate dehydrogenase were shown to have elevated levels of citrate and isocitrate. However, they showed no phenotype and the levels of 2-OG were not lower than in wild-type plants [160].
如果柠檬酸盐而不是 2-OG 是线粒体输出的主要有机酸(参见第 3.7 节),则胞质溶胶中会产生额外的还原当量(可能需要硝酸盐还原)。这将改变我们对线粒体代谢的理解,并强调胞质 NADP+ 依赖性异柠檬酸脱氢酶的重要性。需要进一步的证据,可能涉及转基因植物,才能证明这一点;例如,没有胞质 NADP+ 依赖性异柠檬酸脱氢酶的转基因植物被证明柠檬酸盐和异柠檬酸盐水平升高。然而,它们没有表现出表型,并且 2-OG 的水平不低于野生型植物 [160]。
A strong interaction between mitochondria and chloroplasts also occurs in the dark, which is demonstrated by the strong response of the reduction state of the dark-adapted PQ pool to respiratory activity. Mitochondrial ATP and reductant are necessary for chloroplast functioning in the dark and to prepare the chloroplast for optimal photosynthetic activity upon re-illumination. This is most obvious under conditions of high light and low temperatures, where ATP is used to maintain the proton gradient across the thylakoid membrane in the dark [132]. Such a condition allows the xanthophyll cycle to offer immediate protection against photoinhibition on re-illumination. Before speculating on the in vivo importance of CR it is essential that the components and especially the PQ-oxidase are isolated and characterised. At this stage the existence and significance of CR remain elusive.
线粒体和叶绿体之间的强烈相互作用也发生在黑暗中,这可以通过暗适应 PQ 池的还原状态对呼吸活动的强烈反应来证明。线粒体 ATP 和还原剂对于叶绿体在黑暗中的功能是必需的,并且对于在重新照明时为叶绿体的最佳光合活性做好准备。这在高光和低温条件下最为明显,其中 ATP 用于在黑暗中维持穿过类囊体膜的质子梯度 [132]。这样的条件允许叶黄素循环在重新照明时提供即时保护,防止光抑制。在推测 CR 的体内重要性之前,必须分离和表征成分,尤其是 PQ-氧化酶。在这个阶段,CR 的存在和意义仍然难以捉摸。

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References

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