莲胚芽叶绿素合成对光照的依赖性

被子植物的叶绿素合成需要光照,但是莲（Nelumbo nucifera Gaertn.)胚芽却一直被猜测具有在黑暗中合成叶绿素的能力,因为莲胚芽变绿是在四重覆盖物（子叶、种皮、果皮和莲蓬）包被下几乎不大可能秀光的环境中发生的,本实验从正反两个方面否定了这种可能性;首先对处于发育早期的莲蓬进行遮光处理。结果发现莲胚芽虽然可以继续发育,但是它的叶绿素合成却受到严重抑制。积累了大量合成叶绿素的前体,并且这些前体主要与依赖光的原叶绿素酸酯氧还酶（LPOR）结合在一起;其次不依赖光的原叶绿素酸酯氧还酶（DPOR）的编码基因在物种间高度保守,但是用PCR的方法在功基因组中却扩增不同源序列,表明莲胚芽不大可能具有在黑暗中合成叶绿素所必需的酶。两方面实验结果表明,莲胚芽的叶绿素合成只能通过依赖光的途径进行。     

Novel Insights into the Enzymology,Regulation and Physiological Functions of Light-dependent Protochlorophyllide Oxidoreductase in Angiosperms

The reduction of protochlorophyllide (Pchlide) is a key regulatory step in the biosynthesis of chlorophyll in phototrophic organisms. Two distinct enzymes catalyze this reduction; a light-dependent NADPH:protochlorophyllide oxidoreductase (POR) and light-independent Pchlide reductase (DPOR). Both enzymes are widely distributed among phototrophic organisms with the exception that only POR is found in angiosperms and only DPOR in anoxygenic photosynthetic bacteria. Consequently, angiosperms become etiolated in the absence of light, since the reduction of Pchlide in angiosperms is solely dependent on POR. In eukaryotic phototrophs, POR is a nuclear-encoded single polypeptide and post-translationally imported into plastids. POR possesses unique features, its light-dependent catalytic activity, accumulation in plastids of dark-grown angiosperms (etioplasts) via binding to its substrate, Pchlide, and cofactor, NADPH, resulting in the formation of prolamellar bodies (PLBs), and rapid degradation after catalysis under subsequent illumination. During the last decade, considerable progress has been made in the study of the gene organization, catalytic mechanism, membrane association, regulation of the gene expression, and physiological function of POR. In this review, we provide a brief overview of DPOR and then summarize the current state of knowledge on the biochemistry and molecular biology of POR mainly in angiosperms. The physiological and evolutional implications of POR are also discussed.     

Activity of the Fe-Branch of Tetrapyrrole Biosynthesis in the Chlorophyll-Deficient Plastome Mutant of Sunflower

The biosynthesis of heme, a plant tetrapyrrole, was studied in the leaves of a chlorophyll-deficient plastome mutant of the sunflower (Helianthus annuus L, line 2-24, albina form). In the light, the content of 5-aminolevulinic acid (ALA) in white mutant leaves was, on the average, ten times less than in that of the wild-type form (line 3629). Chlorophyll content in mutant leaves comprised only 0.3% of that of control plants. The activities of Fe-chelatase and ALA dehydratase in the heme synthesis were either comparable to or even higher than those in the wild-type leaves. A normal respiration rate in white mutant leaves, the equal content of phytochrome apoproteins in plants of both types, and the lack of noticeable morphogenetic differences realized through the phytochrome system can indicate that mutant and wild-type leaves are similar in their levels of phytochrome and the cytochromes of mitochondrial respiration. Nevertheless, in the mutant, the content of heme noncovalently bound by apoproteins amounted to only one third of its content in the wild-type plants. It seems that a dramatic decrease in the capability of white leaves for chlorophyll biosynthesis and for the formation of the photosynthetic apparatus is responsible for a low demand for chloroplast cytochromes, which is the major cause of a reduced heme content in the mutant.     

Biosynthesis of Chlorophyll of Photosystem II Reaction Centers in Plant Leaves in Early Stages of Etiolation

Spectral methods were used to study the sequences of chlorophyll biosynthesis reactions in etiolated pea, bean, and maize plants in early stages (3-4 days) of growth. For these juvenile plants, along with the reaction chain known for mature (7-9 day-old) plants, a new reaction chain was found which started with phototransformation of the long-wavelength form PChld 686/676 into PChld 653/648. (PChld 653/648 differs from the main known precursor form PChld 655/650). The subsequent photoreduction of PChld 653/648 leads to the formation of Chld 684/676, which is transformed into Chl 688/680 in the course of a dark reaction. After completion of this reaction, fast (20-30 sec) quenching of the fluorescence of the reaction product is observed with the formation of non-fluorescent Chl 680. The reaction accompanied by pigment fluorescence quenching is absent in pea mutants with depressed function of Photosystem II reaction centers. This suggests that the newly found reaction chain leads to the formation of chlorophyll of the Photosystem II reaction center.     

Regulation of the carotenoid content and chloroplast development by levulinic acid

Chloroplast development and chlorophyll biosynthesis are co-regulated. To understand the mechanism of regulation of chloroplast biogenesis by chlorophyll, development of the photosynthetic apparatus was monitored during greening of etiolated barley leaf discs in the presence of levulinic acid, an inhibitor of chlorophyll biosynthesis. Although not a direct inhibitor of carotenoid biosynthesis, treatment by levulinic acid resulted in a linear reduction in both chlorophyll and carotenoid contents. Chlorophyll biosynthesis appeared to control that of carotenes. In the presence of levulinic acid, photosystem II (PSII) activity decreased while photosystem I (PSI) activity increased when expressed on a chlorophyll basis. However, the activities of both photosystem I and II decreased when expressed on a per plastid basis. As expected, in the presence of low amounts of chlorophyll, the light-harvesting chlorophyll-protein complex II (LHCPII) was not visible in Coomassie-stained gels in 20 m M levulinic acidtreated tissues, but was detected as a faint band by immunoblotting. This small amount of the LHCPII induced significant amounts of grana stacking, which was monitored as an increase in the ratio of variable to maximum fluorescence. When levulinic acid was washed from the leaf discs and the latter allowed to green in its absence, the chlorophyll and carotenoid contents and the photosynthetic activities approached the control values. Levulinic acid could be used to arrest the light-induced chloroplast development at a desired phase of greening and removed by washing the leaves to restore the developmental process without any apparent toxic effect. Results demonstrate that biosynthesis of carotenes is regulated by that of chlorophylls and extremely low amounts of the LHCPII can induce grana stacking.     

Biosynthesis of Biopterin by Rat Brain

Abstract: A method for the determination of [¹⁴C]biopterin biosynthesis from [¹⁴C]guanosine-5'-triphosphate by a desalted preparation from rat striatum, based on sequential reverse-phase and cation-exchange high performance liquid chromatography, is described. Synthesis of reduced forms of biopterin by this striatal extract was found to be dependent on enzymatic activity, guanosine-5'-triphosphate, magnesium ions, and a reduced pyridine nucleotide. As demonstrated by the technique of isotope dilution, isotope trapping, 6-lactyl-7,8-dihydropterin (sepiapterin) was found to be an intermediate in biopterin biosynthesis that is catalyzed by the striatal extract. Rat brain was also shown to synthesize biopterin in vivo from intraventricularly administered [¹⁴C]guanosine or sepiapterin. Intraventricular injection of sepiapterin increased dihydro- and 5,6,7,8-tetrahydrobiopterin levels in rat brain by more than eightfold. The temporal relationship between the appearance of dihydro- and 5,6,7,8-tetrahydrobiopterin following intraventricular injection of sepiapterin suggests that dihydrobiopterin is the immediate product of sepiapterin reduction which is then reduced further to the functional cofactor 5,6,7,8-tetra-hydrobiopterin. Therefore, in contrast to previous reports, the biosynthesis of biopterin by rat brain does not appear to differ from that occurring in other, nonneural tissues.     

Effect of Ni2+ on Early Stages of Chlorophyll Biosynthesis and Pheophytinization in Euglena gracilis

The effect of Ni²⁺ on the early stages of chlorophyll biosynthesis and pheophytinization in Euglena gracilis cells was studied. Incubation of the cells with 10^–4 M Ni²⁺ for 7 days resulted in a higher chlorophyll content, enhanced production of 5-aminolevulinic acid (ALA), and in increased activity of 5-aminolevuluinic acid dehydratase (EC 4.2.1.24, ALAD), as compared to the control cells incubated without Ni²⁺. At a higher concentration (10^–3 M), Ni²⁺ markedly inhibited chlorophyll accumulation and ALAD activity, as compared to the control cells. At this concentration, Ni²⁺ also inhibited heme biosynthesis and strongly stimulated ALA production. It seems likely that, by affecting heme synthesis, Ni²⁺ increases the activity of the ALA production system. However, the suppression of subsequent stages of ALA conversion to chlorophyll, in particular ALAD inhibition, ultimately resulted in almost complete inhibition of chlorophyll biosynthesis. In addition to cessation of de novo chlorophyll synthesis in the presence of Ni²⁺ (10^–3 M) in Euglena cells, the existing chlorophyll was converted into pheophytin and almost completely degraded. We suppose that the Ni²⁺-induced pheophytinization is caused by an acidic shift of intracellular pH related to an impairment of cell membrane permeability by Ni²⁺ cations.     

The third member of the hemA gene family encoding glutamyl-tRNA reductase is primarily expressed in roots in Hordeum vulgare

Accumulation of chlorophylls and heme is primarily controlled at the level of 5-aminolevulinate (ALA) synthesis in higher plants. ALA is formed from glutamate in three enzymatic steps in plants. Among them, the reduction of glutamyl-tRNA^Gluto glutamate-1-semialdehyde (GSA) is likely to be a regulatory point of ALA synthesis. This reaction is catalyzed by glutamyl-tRNA reductase (GTR), which is encoded by a hemA gene. We have isolated a novel isoform of a hemA cDNA clone from barley (Hordeum vulgare) that is the third member of the hemA gene family. mRNA of this isoform is accumulated primarily in roots, suggesting that the isoform is regulated in an organ-specific manner by the demand for heme synthesis rather than chlorophyll. Phylogenetic analysis was done using the deduced amino acid sequences of hemA isoforms from barley, cucumber and Arabidopsis thaliana. The results indicate that the existing gene families in these plants arose after the divergence of monocotyledonous and dicotyledonous plants.     

植物花色素苷生物合成的转录调控

转录调节是植物花色素苷生物合成途径中调节其结构基因表达的重要环节之一。近十多年来,通过调节转录因子的表达激活或抑制有效地调控植物中花色素苷的合成成了众多植物学家研究的重点。现简要介绍了花色素苷的合成运输过程与液泡的沉积扣押、转录因子与调控及转录调节在遗传改良中的应用等方面的研究进展。     

Heme biosynthesis in mammalian systems: evidence of a Schiff base linkage between the pyridoxal 5'-phosphate cofactor and a lysine residue in 5-aminolevulinate synthase.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in nonplant higher eukaryotes. Murine erythroid 5-aminolevulinate synthase has been purified to homogeneity from an Escherichia coli overproducing strain, and the catalytic and spectroscopic properties of this recombinant enzyme were compared with those from nonrecombinant sources (Ferreira, G.C. & Dailey, H.A., 1993, J. Biol. Chem. 268, 584-590). 5-Aminolevulinate synthase is a pyridoxal 5'-phosphate-dependent enzyme and is functional as a homodimer. The recombinant 5-aminolevulinate synthase holoenzyme was reduced with tritiated sodium borohydride and digested with trypsin. A single peptide contained the majority of the label. The tritiated peptide was isolated, and its amino acid sequence was determined; it corresponded to 15 amino acids around lysine 313, to which pyridoxal 5'-phosphate is bound. Significantly, the pyridoxyllysine peptide is conserved in all known cDNA-derived 5-aminolevulinate synthase sequences and is present in the C-terminal (catalytic) domain. Mutagenesis of the 5-aminolevulinate synthase residue, which is involved in the Schiff base linkage with pyridoxal 5'-phosphate, from lysine to alanine or histidine abolished enzyme activity in the expressed protein.     

高等植物赤霉素生物合成及其调节研究进展

主要介绍近年来高等植物中生物活性GAs的生物合成,拟南芥GA生物合成途径中关键酶基因（GA1－GA5）的克隆和GA3基因CYP701A3的母（Saccharomyces cerevisiae）中的成功表达。评述了活性GAs对赤霉不生物合成的反馈抑制作用和反馈调节中信号的传递和接收问题。高等植物中光周期对GA生物合成的调节主要是在20－氧化和／或3β－羟基化步骤。     

植物原花青素生物合成及调控研究进展

原花青素是通过类黄酮途径生成的一类多酚类化合物.原花青素具有重要的生物学功能,不仅是植物应对生物和非生物胁迫的一种重要防御手段,还能影响植物外观、风味和品质,因此原花青素合成途径一直是作物性状改良的研究热点.该文主要在模式植物拟南芥研究的基础上,综述了原花青素生物合成研究的最新进展,讨论了原花青素遗传工程应用前景和主要...     

冠毒素生物合成与调控的研究进展

冠毒素是80年代末发现的一种能引起多种植物弥散性黄萎病细菌毒素,它在结构和功能上与在胁迫反应中起作用的植物内源激素茉莉酸甲酯有显著的相似性。本文阐述了冠毒素在生物合成途径与遗传学研究及生物合成调控机制方面的研究进展。     

POR C of Arabidopsis thaliana: a third light- and NADPH-dependent protochlorophyllide oxidoreductase that is differentially regulated by light

During the sequencing of the genome of Arabidopsis thaliana a gene has been identified that encodes a novel NADPH-protochlorophyllide oxidoreductase (POR)-like protein (accession number AC 002560). This protein has been named POR C. We have expressed the POR C protein in Escherichia coli and have determined its in vitro activity. POR C shows the characteristics of a light-dependent and NADPH-requiring POR similar to POR A and POR B. The expression of the POR C gene differs markedly from that of the POR A and POR B genes. In contrast to the POR A and POR B mRNAs, the POR C mRNA has been shown previously to accumulate only after the beginning of illumination. In light-adapted mature plants only POR B and POR C mRNAs were detectable. The amounts of both mRNAs show pronounced diurnal rhythmic fluctuations. While the oscillations of POR B mRNA are under the control of the circadian clock, those of POR C mRNA are not. Another difference between POR B and POR C was found in seedlings that were grown under continuous white light. The concentration of POR C mRNA rapidly declined and soon dropped beyond the limit of detection, after these seedlings were transferred to the dark. On the other hand, POR B mRNA was unaffected by this light/dark shift. When seedlings were exposed to different light intensities, the amounts of POR B mRNA remained the same, while POR A and POR C mRNAs were modulated in an inverse way by these light intensity changes. POR A mRNA was still detectable in seedlings grown under low light intensities but disappeared at higher light intensities, while the mRNA concentration of POR C rose with increasing light intensities. These different responses to light suggest that the functions of the three PORs of Arabidopsis are not completely redundant, but may allow the plant to adapt its needs for chlorophyll biosynthesis more selectively by using preferentially one of the three enzymes under a given light regime.     

茉莉酸生物合成的调控及其信号通路

茉莉酸类化合物作为一种细胞信号分子,在植物的生长发育、机械损伤、代谢调节及诱导防御相关基因表达等方面起着重要的作用。本文概述了茉莉酸的生物合成调控以及人们目前对茉莉酸信号通路的认识,并对该研究领域存在的问题及今后可能的研究方向进行展望。     

Chloroplast culture: The chlorophyll repair potential of mature chloroplasts incubated in a simple medium

The chlorophyll repair potential of mature Cucumis chloroplasts incubated in a simple Tris-HCI/sucrose medium is described. The chloroplasts were isolated from green, fully expanded Cucumis cotyledons which were capable of chlorophyll repair. This was evidenced by a functional chlorophyll biosynthetic pathway in the mature tissue. The biosynthesis of protochlorophyllide from exogenous δ-aminolevulinic acid was used as a marker for the operation of the chlorophyll biosynthetic chain between δ-aminolevulinic acid and protochlorophyllide. The conversion of exogenous protochlorophyllide into chlorophyll a was used as a marker for the operation of the chlorophyll pathway beyond protochlorophyllide. It appeared from these studies that contrary to published reports, unfortified fully developed Cucumis chloroplasts incubated in Tris-HCl/sucrose without the addition of cofactors exhibited a partial and limited chlorophyll repair capability. Their net tetrapyrrole biosynthetic competence from δ-aminolevulinic acid was confined to the accumulation of coproporphyrin. No net tetrapyrrole biosynthesis beyond coproporphyrin was observed. However, the plastids were capable of incorporating small amounts of δ-amino-[4-¹⁴C]levulinic acid into [¹⁴C] protochlorophyllide but were incapable of converting exogenous protochlorophyllide into chlorophyll. After prolonged incubation of the unfortified chloroplasts in the dark, a fluorescent protochlorophyllide-like compound accumulated. This compound [Cp (E₄₃₀-F₆₃₁)] exhibited a soret excitation maximum at 430 nm (E₄₃₀) and a fluorescence emission maximum at 631 nm (F₆₃₁) in methanol/acetone (4 : 1, v/v). Cp (E₄₃₀-F₆₃₁) was shown to be neither protochlorophyllide nor zinc-protochlorophyllide but an enzymatic degradation product of chlorophyll. The exact chemical identity of this compound has not yet been determined.     

植物叶绿素降解途径及其分子调控

文章介绍了近年来在叶绿素降解产物结构解析和关键酶基因克隆方面的最新成果,以及在此基础上的叶绿素降解途径修正及其分子调控机理研究。     

植物萜类次生代谢及其调控

植物次生代谢在植物生长发育、环境适应、抵御病虫害等方面发挥着重要作用,这些天然产物组成地球上最丰富的有机化合物的宝库．萜类是植物代谢产物中种类最多的一类,具有重要的生理和生态功能,一些成分还有应用价值．近十几年来,人们在萜类化合物的分离、鉴定、应用、生物合成、相关基因与基因族、酶蛋白结构和功能、代谢调控以及代谢工程等各方面取得了重大进展．本文概述了植物萜类化合物代谢及其调控领域的研究进展与发展趋势．     

支链氨基酸合成调控机制及菌种选育策略研究进展

提高国内支链氨基酸产生菌的高产菌株选育水平有助于缩短与国外生产之间的差距,满足国内市场需求。根据支链氨基酸生物合成途径及代谢调节,重点阐述了合成过程中关键酶的代谢调控,介绍了诱变育种、代谢工程、基因组改组及全局转录机器工程四种育种策略的研究进展。在支链氨基酸选育方面,全局转录机器工程育种目前虽无成功实例,但具有很大的潜力,而其他育种策略在氨基酸的选育中均发挥重要作用,可供国内相关育种工作者参考使用。