Chlorophyll Degradation: The Tocopherol Biosynthesis-Related Phytol Hydrolase in Arabidopsis Seeds Is Still Missing

Phytyl diphosphate (PDP) is the prenyl precursor for tocopherol biosynthesis. Based on recent genetic evidence, PDP is supplied to the tocopherol biosynthetic pathway primarily by chlorophyll degradation and sequential phytol phosphorylation. Three enzymes of Arabidopsis (Arabidopsis thaliana) are known to be capable of removing the phytol chain from chlorophyll in vitro: chlorophyllase1 (CLH1), CLH2, and pheophytin pheophorbide hydrolase (PPH), which specifically hydrolyzes pheophytin. While PPH, but not chlorophyllases, is required for in vivo chlorophyll breakdown during Arabidopsis leaf senescence, little is known about the involvement of these phytol-releasing enzymes in tocopherol biosynthesis. To explore the origin of PDP for tocopherol synthesis, seed tocopherol concentrations were determined in Arabidopsis lines engineered for seed-specific overexpression of PPH and in single and multiple mutants in the three genes encoding known dephytylating enzymes. Except for modestly increasing tocopherol content observed in the PPH overexpressor, none of the remaining lines exhibited significantly reduced tocopherol concentrations, suggesting that the known chlorophyll-derived phytol-releasing enzymes do not play major roles in tocopherol biosynthesis. Tocopherol content of seeds from double mutants in NONYELLOWING1 (NYE1) and NYE2, regulators of chlorophyll degradation, had modest reduction compared with wild-type seeds, although mature seeds of the double mutant retained significantly higher chlorophyll levels. These findings suggest that NYEs may play limited roles in regulating an unknown tocopherol biosynthesis-related phytol hydrolase. Meanwhile, seeds of wild-type over-expressing NYE1 had lower tocopherol levels, suggesting that phytol derived from NYE1-dependent chlorophyll degradation probably doesn’t enter tocopherol biosynthesis. Potential routes of chlorophyll degradation are discussed in relation to tocopherol biosynthesis.Vitamin E tocochromanols are lipidic antioxidants found in photosynthetic organisms that exist as two alternate classes, tocopherols and tocotrienols, which differ in the degree of saturation of the hydrophobic C20 prenyl side chain classes. Among these two classes, four forms occur that differ in methylation of the hydrophilic tocochromanol head group (Sattler et al., 2004). The initial step of tocopherol biosynthesis is the condensation of the aromatic head group precursor homogentisate and the prenyl tail precursor phytyl diphosphate (PDP). This reaction is catalyzed by a plastid-localized enzyme, homogentisate PDP transferase (HPT; Soll et al., 1980; Collakova and DellaPenna, 2001). PDP for tocopherol biosynthesis is either provided through direct reduction of geranylgeranyl diphosphate (Keller et al., 1998) or from chlorophyll-bound phytol through chlorophyll hydrolysis and subsequent conversion of free phytol into PDP by two consecutive kinase reactions (Fig. 1; Rise et al., 1989; Goffman et al., 1999; Matile et al., 1999; Kräutler, 2002; Hörtensteiner, 2006). The first of these phosphorylation steps was shown to be catalyzed by vitamin E pathway5 (VTE5; Valentin et al., 2006).Open in a separate windowFigure 1.The substrate PDP directing toward tocopherol biosynthesis is primarily derived from chlorophyll degradation. Two phytol-releasing activities are known, i.e. CLH catalyzing release from chlorophyll and PPH dephytylating pheophytin. Phytol is then converted to PDP by sequential kinase reactions catalyzed by VTE5 and a second, unknown kinase. Condensation of PDP and homogentisate by HPT marks the initial reaction of tocopherol biosynthesis. phy, Phytyl. [See online article for color version of this figure.]Seeds of the Arabidopsis (Arabidopsis thaliana) vte5 mutant have only about 20% of wild-type concentrations of vitamin E, while containing 3-fold more free phytol compared with seeds of wild-type plants (Valentin et al., 2006). In addition, it has been shown that tocopherol accumulation in Brassica napus seeds correlates with chlorophyll breakdown during seed development (Valentin et al., 2006). Therefore, it was concluded that in Arabidopsis, the 80% of PDP that is used for VTE5-dependent tocopherol biosynthesis in seeds arises from free phytol released during chlorophyll degradation. Chlorophyll degradation is an important catabolic process that is catalyzed by a multistep pathway and occurs during leaf senescence and fruit ripening. An early reaction of the chlorophyll degradation pathway is dephytylation. The true identity of the enzyme(s) associated with phytol release has only recently been revealed. It was long believed that chlorophyllase (CLH) is responsible for phytyl hydrolysis, yielding chlorophyllide and free phytol (Heaton and Marangoni, 1996; Takamiya et al., 2000; Hörtensteiner, 2006). However, analysis of the two CLHs in Arabidopsis, AtCLH1 and AtCLH2 (Tsuchiya et al., 1999; Takamiya et al., 2000), indicated that the AtCLH isoforms are neither chloroplast localized nor essential for senescence-related chlorophyll breakdown (Schenk et al., 2007). These findings are consistent with the observation that not all molecularly identified CLHs contain a predicted chloroplast transit peptide (Jacob-Wilk et al., 1999; Tsuchiya et al., 1999). As a consequence, subcellular compartments distinct from plastids were considered to be additional sites of chlorophyll degradation (Takamiya et al., 2000). By contrast, results obtained from Citrus spp. suggested that CLH functions as a rate-limiting enzyme in chlorophyll catabolism within the chloroplast and is controlled by posttranslational regulation (Harpaz-Saad et al., 2007; Azoulay Shemer et al., 2008). Additionally, work in Arabidopsis indicated that clh2 mutants showed a slight delay in chlorophyll degradation compared with clh1 and wild-type plants (Schenk et al., 2007).More recently, a novel plastid-localized enzyme, pheophytin pheophorbide hydrolase (PPH), was shown to be essential for chlorophyll breakdown during leaf senescence in Arabidopsis. PPH catalyzes the dephytylation of pheophytin rather than chlorophyll, resulting in pheophorbide and free phytol as the products (Schelbert et al., 2009). pph mutants are unable to degrade chlorophyll during senescence and therefore exhibit a stay-green phenotype in leaves. Altogether, these data reflect the complexity of the process of chlorophyll dephytylation and raise the question whether any of these activities may be related to tocopherol biosynthesis.Recently, Gregor Mendel’s green cotyledon gene stay-green (SGR), encoding a chloroplast-localized protein, was shown to be required for the initiation of chlorophyll breakdown (Armstead et al., 2007; Sato et al., 2007). Like in many plant species (Hörtensteiner, 2009), NON-YELLOWING1 (NYE1; also named SGR1), the Arabidopsis homolog of SGR, plays an important positive regulatory role in chlorophyll degradation during senescence, because NYE1 overexpression resulted in either pale-yellow leaves or even albino seedlings, while nye1 mutants retain chlorophyll during senescence (Ren et al., 2007). In addition, the second isoform of NYE in Arabidopsis, NYE2 (also named SGR2), is a negative regulator of chlorophyll degradation in senescent leaves (Sakuraba et al., 2014). By contrast, both enzymes positively contribute to chlorophyll breakdown during seed maturation (Delmas et al., 2013). NYE1 and NYE2 were shown to interact at light-harvesting complex II (LHCII) with other chlorophyll catabolic enzymes, including PPH. This suggests that SGRs might function as scaffold proteins in the formation of a catabolic multienzyme complex regulating chlorophyll degradation (Sakuraba et al., 2012, 2014). Whether NYE1 and NYE2 may also affect CLH function remains unclear, but their role as a key regulators for chlorophyll degradation raises the question whether NYEs may also play a role in tocopherol biosynthesis.Here, by employing Arabidopsis transferred DNA (T-DNA) insertion or nonsense mutants that are defective in known chlorophyll degradation-associated genes, and by PPH or NYE1 overexpression, we provide genetic and physiological evidence that neither CLHs nor PPH plays a major role in tocopherol biosynthesis in Arabidopsis seeds.     

突尼斯非洲跳鼠（Jaculus jaculus）和埃及跳鼠J.orientalis）群体的等位酶多态及遗传分化

运用16种酶蛋白编码的23个遗传座位对突尼斯非洲跳鼠(Jaculus jaculus)和埃及跳鼠(J.orientalis)自然群体的遗传变异和分化进行了电泳分析.结果表明,与其他啮齿动物等哺乳动物的相关数据比较,发现这两个种群体的遗传变异水平较低.非洲跳鼠群体的观测杂合度(Hobs)为0.08-0.19,多态座位百分比(P)为26.2%-45.2%,每个座何的平均等位基因数(A)为1.1-1.4;埃及跳鼠的Hobs为0.10-0.15,P为29.3%-44.1%,A为1.1-1.7.两个种群体各自的遗传分化程度较低(非洲跳鼠和埃及跳鼠的Fst分别为0.0017和0.0019).而两个种群体间的Fst为0.607(P＜0.05),表明两个种之间高度的遗传分化.本研究支持这两个种分类地位的合法性,并强调了地理因素(环境类犁和生物气候阶段)对两个种遗传结构的影响.     

Structural Basis for Evolution of Product Diversity in Soybean Glutathione Biosynthesis

The redox active peptide glutathione is ubiquitous in nature, but some plants also synthesize glutathione analogs in response to environmental stresses. To understand the evolution of chemical diversity in the closely related enzymes homoglutathione synthetase (hGS) and glutathione synthetase (GS), we determined the structures of soybean (Glycine max) hGS in three states: apoenzyme, bound to γ-glutamylcysteine (γEC), and with hGSH, ADP, and a sulfate ion bound in the active site. Domain movements and rearrangement of active site loops change the structure from an open active site form (apoenzyme and γEC complex) to a closed active site form (hGSH•ADP•SO₄²⁻ complex). The structure of hGS shows that two amino acid differences in an active site loop provide extra space to accommodate the longer β-Ala moiety of hGSH in comparison to the glycinyl group of glutathione. Mutation of either Leu-487 or Pro-488 to an Ala improves catalytic efficiency using Gly, but a double mutation (L487A/P488A) is required to convert the substrate preference of hGS from β-Ala to Gly. These structures, combined with site-directed mutagenesis, reveal the molecular changes that define the substrate preference of hGS, explain the product diversity within evolutionarily related GS-like enzymes, and reinforce the critical role of active site loops in the adaptation and diversification of enzyme function.     

A reliable measure of similarity based on dependency for short time series: an application to gene expression networks

Background  

Microarray techniques have become an important tool to the investigation of genetic relationships and the assignment of different phenotypes. Since microarrays are still very expensive, most of the experiments are performed with small samples. This paper introduces a method to quantify dependency between data series composed of few sample points. The method is used to construct gene co-expression subnetworks of highly significant edges.     

Sphingolipid C-9 Methyltransferases Are Important for Growth and Virulence but Not for Sensitivity to Antifungal Plant Defensins in Fusarium graminearum

The C-9-methylated glucosylceramides (GlcCers) are sphingolipids unique to fungi. They play important roles in fungal growth and pathogenesis, and they act as receptors for some antifungal plant defensins. We have identified two genes, FgMT1 and FgMT2, that each encode a putative sphingolipid C-9 methyltransferase (C-9-MT) in the fungal pathogen Fusarium graminearum and complement a Pichia pastoris C-9-MT-null mutant. The ΔFgmt1 mutant produced C-9-methylated GlcCer like the wild-type strain, PH-1, whereas the ΔFgmt2 mutant produced 65 to 75% nonmethylated and 25 to 35% methylated GlcCer. No ΔFgmt1ΔFgmt2 double-knockout mutant producing only nonmethylated GlcCer could be recovered, suggesting that perhaps C-9-MTs are essential in this pathogen. This is in contrast to the nonessential nature of this enzyme in the unicellular fungus P. pastoris. The ΔFgmt2 mutant exhibited severe growth defects and produced abnormal conidia, while the ΔFgmt1 mutant grew like the wild-type strain, PH-1, under the conditions tested. The ΔFgmt2 mutant also exhibited drastically reduced disease symptoms in wheat and much-delayed disease symptoms in Arabidopsis thaliana. Surprisingly, the ΔFgmt2 mutant was less virulent on different host plants tested than the previously characterized ΔFggcs1 mutant, which lacks GlcCer synthase activity and produces no GlcCer at all. Moreover, the ΔFgmt1 and ΔFgmt2 mutants, as well as the P. pastoris strain in which the C-9-MT gene was deleted, retained sensitivity to the antifungal plant defensins MsDef1 and RsAFP2, indicating that the C-9 methyl group is not a critical structural feature of the GlcCer receptor required for the antifungal action of plant defensins.     

Mining the bitter melon (<Emphasis Type="Italic">momordica charantia</Emphasis>l.) seed transcriptome by 454 analysis of non-normalized and normalized cDNA populations for conjugated fatty acid metabolism-related genes

Background  

Seeds of Momordica charantia (bitter melon) produce high levels of eleostearic acid, an unusual conjugated fatty acid with industrial value. Deep sequencing of non-normalized and normalized cDNAs from developing bitter melon seeds was conducted to uncover key genes required for biotechnological transfer of conjugated fatty acid production to existing oilseed crops. It is expected that these studies will also provide basic information regarding the metabolism of other high-value novel fatty acids.     

Defining the role of phosphomethylethanolamine N-methyltransferase from Caenorhabditis elegans in phosphocholine biosynthesis by biochemical and kinetic analysis

In plants and Plasmodium falciparum, the synthesis of phosphatidylcholine requires the conversion of phosphoethanolamine to phosphocholine by phosphoethanolamine methyltransferase (PEAMT). This pathway differs from the metabolic route of phosphatidylcholine synthesis used in mammals and, on the basis of bioinformatics, was postulated to function in the nematode Caenorhabditis elegans. Here we describe the cloning and biochemical characterization of a PEAMT from C. elegans (gene, pmt-2; protein, PMT-2). Although similar in size to the PEAMT from plants, which contain two tandem methyltransferase domains, PMT-2 retains only the C-terminal methyltransferase domain. RNA-mediated interference experiments in C. elegans show that PMT-2 is essential for worm viability and that choline supplementation rescues the RNAi-generated phenotype. Unlike the plant and Plasmodium PEAMT, which catalyze all three methylations in the pathway, PMT-2 catalyzes only the last two steps in the pathway, i.e., the methylation of phosphomonomethylethanolamine (P-MME) to phosphodimethylethanolamine (P-DME) and of P-DME to phosphocholine. Analysis of initial velocity patterns suggests a random sequential kinetic mechanism for PMT-2. Product inhibition by S-adenosylhomocysteine was competitive versus S-adenosylmethionine and noncompetitive versus P-DME, consistent with formation of a dead-end complex. Inhibition by phosphocholine was competitive versus each substrate. Fluorescence titrations show that all substrates and products bind to the free enzyme. The biochemical data are consistent with a random sequential kinetic mechanism for PMT-2. This work provides a kinetic basis for additional studies on the reaction mechanism of PEAMT. Our results indicate that nematodes also use the PEAMT pathway for phosphatidylcholine biosynthesis. If the essential role of PMT-2 in C. elegans is conserved in parasitic nematodes of mammals and plants, then inhibition of the PEAMT pathway may be a viable approach for targeting these parasites with compounds of medicinal or agronomic value.     

Effects of 3-beta-diol, an androgen metabolite with intrinsic estrogen-like effects, in modulating the aquaporin-9 expression in the rat efferent ductules

Background  

Fluid homeostasis is critical for normal function of the male reproductive tract and aquaporins (AQP) play an important role in maintenance of this water and ion balance. Several AQPs have been identified in the male, but their regulation is not fully comprehended. Hormonal regulation of AQPs appears to be dependent on the steroid in the reproductive tract region. AQP9 displays unique hormonal regulation in the efferent ductules and epididymis, as it is regulated by both estrogen and dihydrotestosterone (DHT) in the efferent ductules, but only by DHT in the initial segment epididymis. Recent data have shown that a metabolite of DHT, 5-alpha-androstane-3-beta-17-beta-diol (3-beta-diol), once considered inactive, is also present in high concentrations in the male and indeed has biological activity. 3-beta-diol does not bind to the androgen receptor, but rather to estrogen receptors ER-alpha and ER-beta, with higher affinity for ER-beta. The existence of this estrogenic DHT metabolite has raised the possibility that estradiol may not be the only estrogen to play a major role in the male reproductive system. Considering that both ER-alpha and ER-beta are highly expressed in efferent ductules, we hypothesized that the DHT regulation of AQP9 could be due to the 3-beta-diol metabolite.     

The essential nature of sphingolipids in plants as revealed by the functional identification and characterization of the Arabidopsis LCB1 subunit of serine palmitoyltransferase

Serine palmitoyltransferase (SPT) catalyzes the first step of sphingolipid biosynthesis. In yeast and mammalian cells, SPT is a heterodimer that consists of LCB1 and LCB2 subunits, which together form the active site of this enzyme. We show that the predicted gene for Arabidopsis thaliana LCB1 encodes a genuine subunit of SPT that rescues the sphingolipid long-chain base auxotrophy of Saccharomyces cerevisiae SPT mutants when coexpressed with Arabidopsis LCB2. In addition, homozygous T-DNA insertion mutants for At LCB1 were not recoverable, but viability was restored by complementation with the wild-type At LCB1 gene. Furthermore, partial RNA interference (RNAi) suppression of At LCB1 expression was accompanied by a marked reduction in plant size that resulted primarily from reduced cell expansion. Sphingolipid content on a weight basis was not changed significantly in the RNAi suppression plants, suggesting that plants compensate for the downregulation of sphingolipid synthesis by reduced growth. At LCB1 RNAi suppression plants also displayed altered leaf morphology and increases in relative amounts of saturated sphingolipid long-chain bases. These results demonstrate that plant SPT is a heteromeric enzyme and that sphingolipids are essential components of plant cells and contribute to growth and development.     

Comprehensive analysis of the Brassica juncea root proteome in response to cadmium exposure by complementary proteomic approaches

Indian mustard (Brassica juncea L.) is known to both accumulate and tolerate high levels of heavy metals from polluted soils. To gain a comprehensive understanding of the effect of cadmium (Cd) treatment on B. juncea roots, two quantitative proteomics approaches – fluorescence two‐dimensional difference gel electrophoresis (2‐D DIGE) and multiplexed isobaric tagging technology (iTRAQ) – were implemented. Several proteins involved in sulfur assimilation, redox homeostasis, and xenobiotic detoxification were found to be up‐regulated. Multiple proteins involved in protein synthesis and processing were down‐regulated. While the two proteomics approaches identified different sets of proteins, the proteins identified in both datasets are involved in similar biological processes. We show that 2‐D DIGE and iTRAQ results are complementary, that the data obtained independently using the two techniques validate one another, and that the quality of iTRAQ results depends on both the number of biological replicates and the number of sample injections. This study determined the involvement of enzymes such as peptide methionine sulfoxide reductase and 2‐nitropropane dioxygenase in alternatives redox‐regulation mechanisms, as well as O‐acetylserine sulfhydrylase, glutathione‐S‐transferase and glutathione‐conjugate membrane transporter, as essential players in the Cd hyperaccumation and tolerance of B. juncea.