高等植物叶绿素降解的PaO途径
田风霞1，王玮2,
1南阳师范学院生命科学学院，河南南阳473061；2作物生物学国家重点实验室/山东农业大学生命科学学院，山东泰安271018
摘 要：文章介绍植物体内叶绿素降解PAO途径研究进展。
关键词：叶绿素降解；滞绿突变；衰老
PaO passway of chlorophyll breakdown in higher plants
TIAN Feng-Xia1, ZANG Jian-Lei1, WANG Wei2,*
1College of Life Sciences, Nanyang Normal University, Nanyang, 473061, China; 2State Key Laboratory of Crop Science/College of Life Sciences, Shandong Agricultural University, Tai’an, 271018, China
Abstract: This paper introduced the research progress of PAO passway of chlorophyll breakdown in higher plants.
Keywords: Chlorophyll degradation; Stay-green Mutant; Senescence
叶绿素(Chl)，地球上最丰富的色素，是用于吸收光能进行光合作用的一个重要组成部分。然而，由于其吸收光能的特性，叶绿素也是个危险分子和潜在的细胞毒素。这种情况发生在植物的光合机构被过度激活的时候，例如在强光条件下，吸收的能量可以被转移到氧，导致活性氧(ROS)的产生。同样，叶绿素的生物合成或降解被抑制也可导致ROS的产生和细胞死亡。在叶片衰老过程中Chl不断被分解，原有的类胡萝卜素致使叶片呈黄色而黄化，这是一种从衰老的组织中回收营养的主动过程[1]。正常生长发育的植物中，大部分Chl存在于叶片的蛋白质复合体中，以游离态形式存在的Chl会对细胞造成光氧化损伤。为了避免游离态Chl及其有色代谢产物对细胞造成光氧化伤害，植物细胞必须快速降解这些物质。
因此，叶绿素的代谢是植物发育过程需要精确调节的过程。叶绿色的降解主要发生在叶片衰老和果实成熟过程中，虽然叶绿素的生物化学和合成调控被广泛深入的研究。但是它对于许多生物和非生物胁迫的响应，仍然不是很理解。叶绿本研究由国家自然科学基金(30671259)资助。
* 通讯作者(Corresponding author):王玮，E-mail: wangw@sdau.edu.cn
第一作者联系方式: E-mail: tianwenxiu1984@163.com
素作为自然界最重要的色素之一，其生物合成途径已被研究的相当仔细，而对其降解过程却知之甚少多年来，叶绿素的降解被认为是一个生物学之谜。
1912年Arthur stoll发现叶绿素酶以来，伴随叶绿素的消失，没有发现相应的代谢产物积累，这也许就是为什么叶绿素降解长期被忽视的原因。我们现在知道，这些降解产物没有被发现的原因仅仅是因为它们没有颜色。在最近几年中，对这些没有颜色的代谢产物结构的鉴定，随着降解产物结构分析和酶基因克隆(叶绿素酶、脱镁叶绿酸a单加氧酶和红色叶绿素代谢产物还原酶)的成功，逐渐揭示了高等植物衰老叶片中叶绿素降解途径的奥秘。
叶绿素降解产物的结构解析是了解降解代谢流向的关键。在相当长的一段时间内，人们在衰老的叶片中找不到与叶绿素降解有关的衍生物，重要的转折点是1991年Kräutler等发现了大麦中非荧光叶绿素代谢物(nonfluorescent chlorophyll catabolite from Hordeum vulgare，Hv-NCC-1，图1)。Hv-NCC-1的结构提示人们两点：第一、氧化开环是发生在二氢卟吩大环的α-位，而不是先前所认为的δ-位；第二、降解物由叶绿素a衍生而来，因此叶绿素b在植物体内先转化为叶绿素a，再行降解。
图1 Hv-NCC-1的结构示意图
Fig.1 Structure of Hv-NCC-1[2] [2]
大多数学者认同叶绿素降解的关键是脱镁叶绿酸a单加氧酶(pheophorbide a oxygenase，PaO)催化卟啉开环，并因此将叶绿素降解途径称为PaO途径[3-4]。因为PAO负责为特征的大环开环反应，出现在下游的是FCCs和NCCs[5]。
只有识别和鉴定叶绿素代谢过程中的作为降解的自然产物的关键代谢产物，才能更好的分析高等植物中普遍存在的叶绿素降解途径。叶绿素的降解途径可以分为两部分：Chl的分解代谢反应分2个阶段。前一个阶段是所有植物所共有的，即Chl降解成无色的呈蓝色荧光的中间产物(primary fluorescent chlorophyllcatabolite，pFCC)，这一阶段需要4种酶。首先了，Chl被叶绿素酶催化脱去植醇基形成脱植基叶绿素a，然后由脱镁螯合酶去除镁离子形成脱镁叶绿
酸a[6]，接下来脱镁叶绿酸a经过由脱镁叶绿酸a加氧酶和红色叶绿素代谢产物还原酶催化的两步反应转化成pFCC，中间过程形成一种不稳定的中间产物红色叶绿素代谢产物(red chlorophyll catabolite，RCC)。最后，pFCC经过几次修饰之后运输至液泡中。在第二个阶段，经过修饰的pFCC在液泡中发生非酶学异构形成最终的非荧光叶绿素代谢产物NCC，这是在液泡的酸性pH条件下进行物种特异性修饰的过程，最后转化形成单吡咯氧化降解产物。将叶绿素代谢产物从衰老的叶绿体转运到液泡是由激活的转运途径来完成的。
本文综述了叶绿素的降解PaO途径研究进展。
1 叶绿素和含氯色素的反应
1.1 叶绿素b到叶绿素a的转变
叶绿素a (chlorophyll a，Chl a)和叶绿素b (chlorophyll b, Chl b)是植物体内最为主要的两种叶绿素，Chl b向Chl a的转化可能是叶绿素降解途径的一部分[7]。虽然有报道表明，植物叶片衰老过程中有脱植基叶绿素b (chlorophyllide b，chlide b)的产生，但没有叶绿素b进一步降解产物的形成[8]。分析大麦叶绿素无色无荧光降解产物(non-fluorescent Chl catabolite，Hv-NCC-1)的结构表明，它与叶绿素a脱植基后的结构相似；此外，存在于液泡中的其他叶绿素降解产物的结构均源自叶绿素a，而非叶绿素b [9]。脱镁叶绿酸a氧化酶(pheophorbide a oxygenase, PaO)的研究表明，脱镁叶绿酸a (pheophorbide a，pheide a)与PaO的亲和力很高，而脱镁叶绿酸b(pheophorbide b，pheide b)对PaO活性则有抑制作用[10]。据此可以推测，Chl b只有在转化为Chl a，后才能进入降解途径。
至今，在已鉴定的叶绿素降解产物中，除了拟南芥的At-NCC-3之外，在C-7位上均含有甲基，即叶绿素a的特征性取代基团[4]，因此叶绿素b向叶绿素a的转化被认为是叶绿素b降解的先决条件。
这一转化过程由两种酶催化完成，它们是NADPH-依赖的叶绿素b还原酶和铁氧还蛋白依赖羟甲基-叶绿素还原酶[11]。叶绿素Chl (ide) b参与叶绿素分解被证实是在衰老过程中增加其活性[12]。最近，Chl(ide) b还原酶可以归因于水稻两个短链脱氢酶/还原酶蛋白，称为NON-YELLOW COLORING1 (NYC1)和NYC1-LIKE (NOL)[13-14]。
2007年Kusaba等[13]在水稻的持绿突变体中，采用图位克隆技术，克隆了NYC1
基因(NON-YELLOW COLORING 1)，结果表明它是编码叶绿体定位并带有3个跨膜结构域的短链脱氢酶/还原酶。数据库检索表明它与水稻和拟南芥中的NOL (NYC1-LIKE)高度相似。研究者将扩增的NOL片段插入pGEX-3X质粒，构建谷胱甘肽S-转移酶的融合蛋白，并在大肠杆菌(BL21 DE3)中表达，细胞抽提物与叶绿素b反应后，采用高效液相色谱分析产物羟甲基-叶绿素，证实了NOL的体外叶绿素b还原酶活性。
1.2 脱植醇
鉴定Pheide a作为叶绿素分解的中间代谢产物[10,15]，表明去除中央镁原子和疏水醇侧链的开环反应优于PaO催化的含氯色素反应环的打开。多年来，chlorophyllase (CLH)催化叶绿素植醇水解反应发生在脱镁之前，即产生Chlide作为中间代谢产物[16–17]，有一些报告认为存在一个相反的顺序，即脱镁发生在脱植醇之前，产生Phein作为中间代谢产物[18,19]。
并不是所有的推断的CLH基因蛋白定位于在叶绿体[20]。此外，最近的拟南芥中的研究表明解决了这个问题。拟南芥中包含两个CLHs，CLH1和CLH2,，但缺失一方或双方各自的突变体蛋白以及RNAi沉默CLH1的植株，只在叶片衰老的有一点边际效应。总的来说，这些数据挑战CLHS在体内叶片的衰老过程中活跃的观点，因此，得出的结论是，在衰老叶绿素降解的过程中CLHs并不是必须需要的[21-22]。
By contrast, several investigations in different species support a function for CLHs. For example, antisense suppression of CLH1 of broccoli (Brassica oleracea) in transgenic plants delayed the rate of postharvest Chl breakdown [74]. Furthermore, Citrus CLH was convincingly shown to be active in Chl breakdown during Citrus fruit ripening, and, after heterologous overexpression in squash leaves and tobacco cells, to also promote Chl breakdown in these leaf tissues [75,76].
相比之下，在不同的物种中的许多调查支持CLHs的功能。例如，对clh1花椰菜(Brassica oleracea)反义抑制在转基因植物中的叶绿素击穿延迟采后[ 74 ]率。此外，柑橘CLH令人信服地证明是活跃在柑橘果实成熟过程中的叶绿素的分解，和异源表达后，南瓜叶和烟草细胞，也促进这些叶组织75,76 ]
A functional genomics screen in Arabidopsis for alternative phytolcleaving esterases uncovered pheophytinase (PPH), a 50 kDa protein located in the chloroplast stroma [14]. Recombinant Arabidopsis PPH exhibited phytol-cleavage activity, which was specific for Phein. Thereby, both Phein a and Phein b were accepted as substrates, but the enzyme did not dephytylate Chl. This intriguing specificity paralleled the fact that Arabidopsis PPH mutants accumulated significant amounts of Phein during senescence, pointing to an in vivo relevance of this enzyme.
[叶绿素分解。一个功能基因组学筛选拟南芥的替代phytolcleaving酯酶发现pheophytinase(PPH)，一个50 kDa蛋白定位于叶绿体基质[ 14 ]。重组拟南芥PPH具有醇裂解活性，它是具体phein。因此，A和B两phein phein接受作为底物，但没有dephytylate叶绿素酶。这个有趣的事实性与拟南芥突变体的大量积累phein PPH衰老过程中，指向一个在体内的这种酶的相关性。
In an independent study [77], the same gene, termed CRN1 (Co-regulated with NYE1), was identified by a reverse genetics approach because of its high co-regulation with NONYELLOWING1 (NYE1), a protein supposedly involved in regulating Chl breakdown (see below). NON-YELLOW COLORING3 (NYC3), a rice (Oryza sativa) gene identified in a mutagenesis screen for retention of greenness during senescence, was shown to be defective in the closest homolog of PPH/CRN1 in rice [78].
在一个独立的研究[ 77 ]，相同的基因，称为CRN1(CO调节NYE1)，确定了反向遗传学的方法，由于其高CO调节nonyellowing1(NYE1)，可能参与调节叶绿素分解蛋白质(见下文)。非coloring3(nyc3)，水稻(Oryza sativa)基因中的突变屏幕衰老过程中保持绿色，被发现在水稻[ 78 ]
The biochemical role of the identified proteins has not been addressed in the latter two studies. Both Arabidopsis PPH/CRN1 and rice NYC3 are serine-type hydrolases that contain a novel consensus domain which is similar to, but distinct from known lipase motifs. A mutation of the active site serine residue of PPH to alanine prevented complementation of the stay-green phenotype of the pph-1 mutant, thereby confirming the in vivo hydrolytic activity of PPH [14]. In summary, PPH/CRN1/NYC3 represents an alternative dephytylating activity. The stay-green phenotype of respective mutants points to an in vivo involvement of these hydrolases in Chl breakdown. It can be concluded that at least in rice and Arabidopsis leaf senescence, dechelation precedes dephytylation, and CLHs are probably not active. Yet for other systems, in particular fruit ripening (see below), involvement of CLHs cannot be ruled out.
PPH / CRN1的最接近的同源基因有缺陷。鉴定的蛋白的生化作用尚未在后者的两个研究。两个拟南芥和水稻nyc3 PPH / CRN1是丝氨酸水解酶，包含一个新的共识的领域是相似的，但不同于已知的脂肪酶的图案。一个突变的PPH的活性位点丝氨酸残基丙氨酸阻止该pph-1突变体的表型保持绿色互补，从而证实了在体内水解活性PPH [ 14 ]。总之，PPH / CRN1 / nyc3代表一种替代dephytylating活动。各突变体的表型分保持绿色的叶绿素分解体内参与这些水解酶。可以得出结论，至少在水稻和拟南芥叶片衰老之前，dechelation dephytylation，和钟可能是不活跃的。然而，其他的系统，特别是果实成熟的(见下文)，参与CLHS不能排除。
1.3 螯合镁
Two types of activities able to catalyze the release of the central Mg atom of Chl have been described in the
literature. Different heat stable low-molecular weight compounds, termed metal chelating substance (MCS), isolated from Chenopodium album (b400 Da) [79] and strawberry (2180 Da) [80] have been shown to catalyze Mg dechelation in vitro. On the other hand, Mg-releasing proteins (MRP) have been described as well [16,81]. Differences between these two activities were shown to reside in their substrate specificity [16,82].
两种类型的活动能够催化叶绿素中心Mg原子释放已在文献中描述。不同的热稳定的低分子量化合物，称为金属螯合物(MCS)，分离从藜(B400 DA)[ 79 ]和草莓(2180 Da)[ 80 ]已被证明是促进体外毫克dechelation。另一方面，Mg释放蛋白(MRP)被描述为[ 16,81 ]。这两种活动之间的差异是居住在他们的底物特异性[ 16,82 ]。
Thus, Mg-dechelatase was active with an artificial Chl substrate, chlorophyllin, but not with the natural substrate, Chlide. In contrast MCS acted on both, indicating that in vivo Mg removal from Chlide most likely is due to the activity of different low molecular weight compounds. The possibility of MCS representing a cofactor of MRP that might easily dissociate from the protein, has been ruled out [83].
因此，镁dechelatase活跃与人工CHL基板，叶绿素，但不与天然底物，聚合。在对比MCS行动，表明从Chlide最有可能是由于去除体内镁不同的低分子量化合物的活性。MCS代表一个辅因子MRP可能容易地从蛋白质分离的可能性，已经排除了[ 83 ]。
Yet, elucidating the molecular nature of MCS is a prerequisite to understand the mechanism of Mg-removal during Chl breakdown. In addition, based on the identification of PPH and the likely possibility that Mg is released from Chl not Chlide, re-examination of the activities of MCS and MRP using Chl as substrate is demanded.
然而，阐明肥大细胞的分子性质是理解Chl击穿Mg去除机理的前提。此外，基于PPH识别和镁是叶绿素不致释放的可能性，对MCS和MRP使用叶绿素作为基板的要求重新检查活动。
3.2. Oxidative ring opening as key reaction
3.2.1. Pheophorbide a oxygenase
Pheide a, the last chlorin-type pigment in Chl breakdown, is oxygenolytically opened by PAO to yield enzyme-bound RCC. PAO is a monooxygenase, which regioselectively attaches an oxygen atom at the C-5 position of Pheide a [22]. PAO is a Rieske-type iron-sulfur oxygenase [29,59] and in Arabidopsis belongs to a five-membered family of Rieske oxygenases [84]. Like other Rieske-type oxygenases, PAO receives electrons from reduced ferredoxin. PAO contains two Cterminally-located transmembrane domains, which were considered to anchor it to the chloroplast envelope [85]. PAO exhibits an intriguing specificity for Pheide a, and Pheide b inhibits the enzyme in a competitive manner [6].
Pheide一，最后二氢卟吩型色素叶绿素分解，由炮产生酶结合的碾压混凝土oxygenolytically打开。炮是
一种单加氧酶，它可以将一个氧原子在Pheide一[ 22 ] C-5位置。炮是Rieske型硫铁氧29,59 ]和[在拟南芥属一五元的家庭的Rieske加氧酶[ 84 ]。像其他的Rieske型氧合酶，减少铁氧还蛋白接收电子报。PAO包含两个cterminally位于跨膜结构域，这被认为是锚定在叶绿体信封[ 85 ]。PAO具有用于Pheide一个有趣的特异性，和Pheide B抑制酶的竞争方式[ 6 ]。
3.2.2. Red chlorophyll catabolite reductase
Attempts to biochemically characterize PAO activity in isolated chloroplast membranes were unsuccessful. Only after the addition of stromal proteins, i.e. a second enzyme fraction, PAO activity could be determined through the production of what is now known as pFCC [6]. The active component of the stroma was identified as RCC reductase (RCCR) and RCC was shown to be an intermediate of the reaction that was not released [20]. Subsequently, physical interaction of PAO and RCCR was demonstrated in a two hybrid screen, indicating that pFCC formation from Pheide a proceeds via metabolic channeling of the intermediary RCC [86].
试图在孤立的叶绿体膜的生化特征的PAO活性都没有成功。只有在基质蛋白的增加，即第二部分PAO活性酶，可以通过什么是现在被称为PFCC [ 6 ]生产确定的。基质的活性成分被确定为RCC还原酶(RCCR)和碾压混凝土被证明是没有被释放的反应中间体[ 20 ]。随后，PAO和RCCR物理相互作用的一二个杂交屏幕显示，表明PFCC形成Pheide一个通过代谢的中间碾压混凝土[ 86 ]窜
RCCR is distantly related to a family of ferredoxin-dependent bilin reductases. These include different bilin reductases from algae and cyanobacteria required for phycobilin synthesis, such as phycocyanobilin:ferredoxin oxidoreductase (PcyA) and phycoerythrobilin synthase (PebS), and phytochromobilin synthase (HY2) of higher plants, catalyzing the final step in phytochrome chromophor synthesis. Like these reductases, RCCR requires ferredoxin as reductant and a radical mechanismas proposed for PcyA and HY2 [87,88] is likely to also occur in RCCR [27].
四是一个家庭的铁氧还蛋白还原酶依赖比林远亲。这些包括从藻类和蓝藻藻胆色素合成所需的不同比林还原酶，如藻蓝素：铁氧还蛋白还原酶(PCya)和藻红素合成酶(PEBS)，和phytochromobilin合酶(2)高等植物光敏色素发色团，催化合成的最后一步。像这些需要铁氧还蛋白还原酶，RCCR作为还原剂和自由基机制提出PCya和HY2 [ 87,88 ]很可能也在发生RCCR [ 27 ]。
The recent elucidation of the crystal structure of Arabidopsis RCCR at 2.4 Å [89] uncovered a high degree of similarity to the structure of PcyA [90] and PebS [91], confirming the catalytic and mechanistic similarity of these reductases. RCCR regio- and stereoselectively reduces the C-20/C-1 double bond of RCC, yielding two possible C-1-stereoisomers, pFCC and epi-pFCC [24]. The source of RCCR defines which isomer is formed and it was shown that mutation of phenylalanine218 to valine in Arabidopsis RCCR changed the stereospecificity of the
pFCC producing Arabidopsis RCCR to epi-pFCC production [86]. Interestingly, phenylalanine218 locates within the putative substrate binding pocket in the crystal structure of RCCR [89].
在2.4Å[ 89 ]的拟南芥RCCR晶体结构最近阐明了相似的PCya [ 90 ]和PEBS [ 91 ]结构高度，确定催化机理的这些酶的相似性。区域选择性和立体选择性降低RCCR RCC的C-20 / C-1双键，产生两个可能的c-1-stereoisomers，PFCC和EPI PFCC [ 24 ]。RCCR源定义形成的异构体的研究表明，在拟南芥phenylalanine218 RCCR缬氨酸突变改变了生产拟南芥RCCR EPI PFCC生产[ 86 ]的PFCC立体定向。有趣的是，phenylalanine218位于假定的底物结合口袋中RCCR [ 89 ]的晶体结构。
3.3. Later transformations of tetrapyrrolic catabolites
The diversity of FCCs and NCCs identified from senescing leaves and ripening fruits of different species demonstrates that after the (common) formation of pFCC in the early pathway described above, additional reactions occur. Overall four peripheral positions are functionalized, i.e. C-3 vinyl, C-8 ethyl, C-132 carboxymethyl and C-17 propionyl (Table 2). In addition, FCCs with a free C-17 propionyl group finally isomerize to their respective NCCs. Some of the reactions, i.e. C-82 hydroxylation and FCC-to-NCC isomerization are commonly found in all species, whereas others occur speciesspecifically The mechanisms involved in these reactions are largely unknown, and only the following reactions have been investigated in recent years.
多样性的设备是从衰老叶片和果实成熟的不同物种的鉴定表明：经过(共同)在上述早期通路PFCC形成，额外的反应。整个四周位置的官能化，即C-3 C-8乙烯，乙酸，c-132羧甲基和C-17丙(表2)。此外，一个免费的C-17丙组最后使各自的控制设备。一些反应，即C-82羟基化和FCC NCC异构化中常见的物种，而其他人发生speciesspecifically机制参与这些反应在很大程度上是未知的，只有以下反应是近年来的研究。