Liver Transcriptome Analysis Reveals Energy Regulation and Functional Impairment of Onychostoma sima During Starvation

Marine Biotechnology - Releasing juvenile fish into resource-depleted waters is regarded as an effective way to restore fishery resources. However, during this stage, released fish are most...     

Phosphate Starvation Inducible Metabolism in Lycopersicon esculentum: II. Characterization of the Phosphate Starvation Inducible-Excreted Acid Phosphatase

Three-day-old suspension cultured cells of Lycopersicon esculentum transferred to a Pi-depleted medium had 2.7 times the excreted acid phosphatase (Apase) activity of cells transferred to a Pi-sufficient medium. Cell growth during this time period was identical for the two treatments. Excreted Apase activity was resolved into two fractions on a Sephadex G-150 column. Most of the phosphate starvation inducible (psi) enhancement in activity was in the lower molecular weight fraction. These two fractions exhibited different substrate versus pH activity profiles. With a native polyacrylamide gel electrophoresis assay, the lower molecular weight fraction resolved into two bands of activity. Both column fractions resolved into the same single band of activity with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weight of this enzyme was 57 kilodalton. These data indicate that L. esculentum has at least two isozymes of the psi-excreted Apase and that these isozymes may associate to form high molecular weight aggregates. Labeling studies using [³⁵S]methionine show that the psi response in tomato cells is complex and involves changes in the steady state levels of several excreted proteins.     

酵母SOD1基因缺失突变体应答刚果红胁迫的转录组学分析

SOD1基因编码的铜锌超氧化物歧化酶是酵母细胞中最重要的抗氧化酶. 前期研究发现,SOD1基因缺失(sod1Δ)导致酵母细胞对真菌细胞壁抑制剂刚果红(Congo red, CR)的敏感性增加,提示细胞抗氧化能力与细胞壁稳定性相关. 本研究采用酵母全基因组表达谱芯片,比较了CR胁迫条件下,野生型酵母细胞和sod1Δ酵母细胞的转录表达谱. 结果表明,与野生型酵母细胞相比,sod1Δ酵母细胞中260个基因发生了显著差异表达(140个基因表达上调、120个基因表达下调). 随机选取12个差异表达基因采用定量PCR验证,结果与芯片分析结果一致. 差异表达基因功能主要涉及细胞壁(几丁质合成)、细胞代谢、细胞防御(抗氧化和热冲击蛋白)、蛋白质合成以及大量功能未知基因. 进一步研究发现,CR处理后,细胞壁几丁质含量和细胞内氧化应激指标丙二醛(MDA)含量在sod1Δ酵母细胞中显著升高,而在野生型酵母细胞中无明显变化,与芯片筛选差异表达基因的生物学功能分析结果一致. 本研究提供了在全基因组水平上对SOD1基因与细胞壁应激反应之间关联的新认识.     

Long-Term Phosphate Starvation and Respiratory Metabolism in Suspension-Cultured Catharanthus roseus Cells

In order to clarify the metabolic adaptation of respiratorypathways in plants to limited levels of P_i, the effects of long-termstarvation of P_i on the activities of various enzymes relatedto respiratory metabolism were examined in suspension-culturedCatharanthus roseus cells. When the activities were expressedas units per g fresh weight, only those of phosphoenolpyruvate-hydrolyzing(PEP-hydrolyzing) enzyme (which may possibly be equivalent tothe acid phosphatase activity derived from vacuoles) and PEPcarboxylase were higher in the Pⁱ-starved cells than in controlcells. Activities of other enzymes in the Pⁱ-starved cells werelower than or similar to those of the control cells. Time-coursestudies indicated that PEP-hydrolyzing activity was inducibleby starvation of Pⁱ. However, in contrast to the results reportedby Duff et al. [(1989a) Plant Physiol. 90: 1275.], fluctuationsin the activity of PP¹:fructose-6-phosphate 1-phosphotransferaseduring starvation of Pⁱ were similar to those in levels of phosphofructokinaseand 6-phosphogluconate dehydrogenase. These data suggest thatthe concept of the phosphate starvation-inducible ‘bypasses’,which are engineered via the coarse control (i.e., induction)of specified enzymes and were proposed initially by Duff etal. in Brassica nigra cells, is not directly applicable to Catharanthusroseus cells in suspension. Tracer experiments using [U-¹⁴C]glutamineindicated that a significant proportion of respiratory substratescould be supplied from the enlarged pool of amino acids duringstarvation of Pⁱ. These assumptions are supported by the observedfluctuations in levels of free amino acids and of protein inP¹-fed and P¹-deficient Catharanthus roseus cells. ¹Part 41 in the series ‘Metabolic Regulation in PlantCell Cultrue’ ²Present Address: Morinaga Mild Industry, 5-1-83, Higashihara,Zamma-shi, Kanagawa, 228 Japan     

Phosphate Starvation Inducible Metabolism in Lycopersicon esculentum: III. Changes in Protein Secretion under Nutrient Stress

Phosphate starvation increased the secretion of at least six proteins by suspension cultured tomato (Lycopersicon esculentum L. and L. pennellii) cells. Cells exhibited a biphasic response to phosphate (Pi) starvation. The early phase involved enhanced secretion of three proteins in response to transfer to a Pi-depleted media, while biomass accumulation continued at the same rate as in the Pi-sufficient cells. Severe starvation, defined as inhibition of biomass accumulation, induced enhanced secretion of three additional proteins. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, media proteins were immunoblotted with antibodies reacting specifically to oligosaccharides processed by the Golgi apparatus. Binding patterns showed that the enhancement in secretion during both phases of starvation was Golgi-mediated. Cells undergoing severe starvation had a respiration rate approximately twice that of unstressed cells and secreted 4.4 times more protein into the media per unit biomass. These data suggest overlapping Pi starvation-specific and global stress responses in plant cells. Under these conditions, Golgi-mediated protein secretion is enhanced. We present evidence for phosphate starvation inducible enhancement of Pi uptake. Secreted proteins specific for N and Fe starvation are also identified.     

Link between Phosphate Starvation and Glycogen Metabolism in Corynebacterium glutamicum,Revealed by Metabolomics

In this study, we analyzed the influence of phosphate (P_i) limitation on the metabolism of Corynebacterium glutamicum. Metabolite analysis by gas chromatography-time-of-flight (GC-TOF) mass spectrometry of cells cultivated in glucose minimal medium revealed a greatly increased maltose level under P_i limitation. As maltose formation could be linked to glycogen metabolism, the cellular glycogen content was determined. Unlike in cells grown under P_i excess, the glycogen level in P_i-limited cells remained high in the stationary phase. Surprisingly, even acetate-grown cells, which do not form glycogen under P_i excess, did so under P_i limitation and also retained it in stationary phase. Expression of pgm and glgC, encoding the first two enzymes of glycogen synthesis, phosphoglucomutase and ADP-glucose pyrophosphorylase, was found to be increased 6- and 3-fold under P_i limitation, respectively. Increased glycogen synthesis together with a decreased glycogen degradation might be responsible for the altered glycogen metabolism. Independent from these experimental results, flux balance analysis suggested that an increased carbon flux to glycogen is a solution for C. glutamicum to adapt carbon metabolism to limited P_i concentrations.Phosphorus is an essential nutrient for all cells and is required for, e.g., the biosynthesis of nucleotides, NAD(P)H, DNA, and RNA but also for the regulation of protein activity by phosphorylation of histidine, aspartate, serine, threonine, or tyrosine residues. A common phosphorus source is inorganic phosphate (P_i), and cells have developed mechanisms for the acquisition, assimilation, and storage of P_i. When P_i becomes limiting, many bacteria induce the synthesis of proteins that enable them to capture the residual P_i resources more efficiently and to make alternative phosphorus sources accessible. The corresponding genes are collectively named P_i starvation-inducible genes, or psi genes. The P_i starvation response, and in particular its regulation, has been most carefully studied in Escherichia coli (45) and Bacillus subtilis (14).We recently started to characterize the P_i starvation response in Corynebacterium glutamicum, a Gram-positive soil bacterium used industrially for the production of more than two millions tons of amino acids per year, mainly l-glutamate and l-lysine (12). An overview of the biology, genetics, physiology, and application of C. glutamicum can be found in two recent monographs (3, 6). Phosphorus constitutes 1.5% to 2.1% of the cell dry weight of C. glutamicum (24), part of which can be present as polyphosphate (22, 29). Several of the enzymes involved in polyphosphate metabolism have been characterized recently, such as a class II polyphosphate kinase (28), the exopolyphosphatases Ppx1 and Ppx2 (26), a polyphosphate/ATP-dependent glucokinase (25), and a polyphosphate/ATP-dependent NAD⁺ kinase (27). The P_i starvation stimulon of C. glutamicum was determined using whole-genome DNA microarrays (15). Comparison of the mRNA profiles before and at different times after a shift from P_i excess to P_i starvation led to the identification of a group of genes that are presumably required to cope with limited P_i supply. This group includes the following: the pstSCAB operon, encoding an ABC transporter for high-affinity P_i uptake; the ugpAEBC operon, encoding an ABC transporter for uptake of glycerol 3-phosphate; glpQ1, encoding a glycerophosphoryl diester phosphodiesterase; ushA, encoding a secreted enzyme with UDP-sugar hydrolase and 5′-nucleotidase activities (33); nucH, encoding a putative secreted nuclease which possibly plays a role in liberating P_i from extracellular nucleic acids; phoC (NCgl2959/cg3393), which may encode a cell wall-associated phosphatase (46); phoH1, encoding an ATPase of unknown function; and the pctABCD operon, encoding an ABC transport system which might be involved in the uptake of a yet-unknown phosphorus-containing compound (15). C. glutamicum lacks homologs of genes for phosphonate degradation, as well as the capability to utilize phosphonates as P sources (15).In most bacteria analyzed in this respect, the P_i starvation response is controlled by two-component signal transduction systems, e.g., the PhoBR system in E. coli (13) and the PhoPR system in B. subtilis (14). Our previous studies revealed that in C. glutamicum, a two-component system composed of the sensor kinase PhoS and the response regulator PhoR is involved in the activation of phosphate starvation-inducible genes (21). Studies with purified proteins showed that phosphorylation by PhoS increased the DNA-binding affinity of PhoR, which bound to many of the P_i starvation-inducible genes, but with different affinities (34).The study reported here was initiated by the question how the metabolism of C. glutamicum responds to P_i limitation. Our results reveal a link between P_i limitation and glycogen metabolism, which was also used for metabolic simulations based on a genome-wide metabolic model.     

Transcriptome Analysis Reveals Candidate Genes Involved in Low Temperature Stress in Bell Pepper

Russian Journal of Plant Physiology - Despite cold stress is a critical environmental condition that influences the growth and development of bell pepper (Capsicum annuum L.), the underlying...     

植物磷饥饿信号与激素信号相互作用

磷酸盐是植物生长、发育、繁殖不可缺少的因子。然而在自然和农业环境中,植物可利用的磷酸盐极为低下,从而提高植物对磷酸盐的利用率至关重要。本文结合近年来国内外的相关研究,就缺磷环境下植物体内相应的信号分子及其相互作用机制进行了阐述。     

Systematic Identification of Regulators of Oxidative Stress Reveals Non-canonical Roles for Peroxisomal Import and the Pentose Phosphate Pathway

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Large Scale Comparative Proteomics of a Chloroplast Clp Protease Mutant Reveals Folding Stress, Altered Protein Homeostasis, and Feedback Regulation of Metabolism

The clpr2-1 mutant is delayed in development due to reduction of the chloroplast ClpPR protease complex. To understand the role of Clp proteases in plastid biogenesis and homeostasis, leaf proteomes of young seedlings of clpr2-1 and wild type were compared using large scale mass spectrometry-based quantification using an LTQ-Orbitrap and spectral counting with significance determined by G-tests. Virtually only chloroplast-localized proteins were significantly affected, indicating that the molecular phenotype was confined to the chloroplast. A comparative chloroplast stromal proteome analysis of fully developed plants was used to complement the data set. Chloroplast unfoldase ClpB3 was strongly up-regulated in both young and mature leaves, suggesting widespread and persistent protein folding stress. The importance of ClpB3 in the clp2-1 mutant was demonstrated by the observation that a CLPR2 and CLPB3 double mutant was seedling-lethal. The observed up-regulation of chloroplast chaperones and protein sorting components further illustrated destabilization of protein homeostasis. Delayed rRNA processing and up-regulation of a chloroplast DEAD box RNA helicase and polynucleotide phosphorylase, but no significant change in accumulation of ribosomal subunits, suggested a bottleneck in ribosome assembly or RNA metabolism. Strong up-regulation of a chloroplast translational regulator TypA/BipA GTPase suggested a specific response in plastid gene expression to the distorted homeostasis. The stromal proteases PreP1,2 were up-regulated, likely constituting compensation for reduced Clp protease activity and possibly shared substrates between the ClpP and PreP protease systems. The thylakoid photosynthetic apparatus was decreased in the seedlings, whereas several structural thylakoid-associated plastoglobular proteins were strongly up-regulated. Two thylakoid-associated reductases involved in isoprenoid and chlorophyll synthesis were up-regulated reflecting feedback from rate-limiting photosynthetic electron transport. We discuss the quantitative proteomics data and the role of Clp proteolysis using a “systems view” of chloroplast homeostasis and metabolism and provide testable hypotheses and putative substrates to further determine the significance of Clp-driven proteolysis.Intracellular proteolysis is important for regulation of metabolic and signaling pathways as well as protein homeostasis and viability of cells and organelles. Chloroplasts contain multiple soluble and membrane-bound proteases and processing peptidases (1) presumably with partially overlapping substrates. These include stromal processing peptidase (2) and stromal PreP1,2 involved in degradation of cleaved transit peptides (3); various amino peptidases (4, 5); the thylakoid processing peptidases cTPA (6), TPP (7), and thylakoid/envelope signal peptidase I (8); and thylakoid-bound proteases SppA (9) and Egy1 (10) as well as stromal and thylakoid members of the Deg, FtsH, and Clp families (11–13). Together with several chaperone systems, including CPN60/CPN10, HSP70/DnaJ, HSP90, and ClpB3 (14), these proteases are part of the chloroplast protein homeostasis network. Importantly the connectivity and overlap of proteins within this homeostasis network is poorly understood; in particular it is unclear how protease substrates are recognized by the different proteolytic systems. Several suppressors of variegated FtsH protease mutants in Arabidopsis have elegantly demonstrated that the balance between protein synthesis and degradation plays an important role in chloroplast homeostasis (15–17). Comparative proteome analysis of chloroplast homeostasis mutants will provide insights in this homeostasis network as we recently showed for a protein sorting mutant (18), and it will identify candidate protease substrates.The Clp proteins form the largest plastid localized protease family with five serine-type ClpP (P1,P3–6) proteases, four non-catalytic ClpR (R1–4) proteins, three Clp AAA⁺ chaperones (C1,C2, and D), and several additional members (ClpT1,T2,S) with unknown functions (1, 13, 19). We note that we renamed Arabidopsis ClpS1,S2 and ClpT as ClpT1,T2 and ClpS to be consistent with the Escherichia coli nomenclature for ClpS (20). The ClpR proteins lack the three catalytic amino acid residues that are conserved across ClpP proteins (21). All proteins of the Arabidopsis Clp proteolytic system have been identified by mass spectrometry (13), including a potential substrate affinity regulator, ClpS.¹Recent evidence shows that the Clp proteolytic system plays a critical role in plant growth, development, and protein homeostasis. ClpP1 is plastid-encoded and was shown to be essential for shoot development in tobacco (22, 23). Down-regulation of the plastid-encoded CLPP1 gene in the green algae Chlamydomonas reinhardtii suggested that ClpP1 is involved in the degradation of the thylakoid-bound subunits of cytochrome b₆f and photosystem II (PSII)² complex (24, 25). Arabidopsis mutant clpr1-1 carries a premature stop codon in the CLPR1 gene and showed a virescent phenotype and delayed chloroplast development and differentiation (26). Maturation of 23 and 4.5 S chloroplast ribosomal RNA (rRNA) is delayed in clpr1-1 (26), but it is not clear how this is related to the loss of ClpR1 protein. Phenotypes of Arabidopsis antisense lines against CLPP4 (27) and CLPP6 (28) also showed delayed chloroplast and plant development as well as reduced accumulation of other ClpP,R subunits. Based on two-dimensional gel analysis, several chloroplast proteins were suggested to be substrates of the Clp machinery (28–30), but direct evidence is lacking. A null mutant for the CLPC1 chaperone (also named HSP93-V) resulted in reduced plant growth, chloroplast development, and protein import rates, but homozygous plants are autotrophic and seeds are viable (31–33). A null mutant for chaperone CLPC2 has no visible phenotype, whereas lack of both CLPC1 and CLPC2 prevents embryogenesis (34). Interestingly ClpC1 is also involved in accumulation of chlorophyll a oxygenase, which is responsible for conversion of chlorophyll a to chlorophyll b (35).In a previous study, we identified and characterized a T-DNA-tagged Arabidopsis thaliana mutant with reduced expression of CLPR2; this mutant was named clpr2-1 (36). Accumulation of the assembled 325-kDa ClpPRT complex was 2–3-fold reduced and resulted in delayed chloroplast and plant development with small chloroplasts and a pale green phenotype. The clpr2-1 mutant shows the strongest visible phenotype when seedlings are young. To better understand the role of the Clp machinery in chloroplast biogenesis and homeostasis and to discover potential Clp substrates, a comprehensive proteome analysis at different points in leaf development of the clpr2-1 mutant is presented in the current study. The methods to quantitatively analyze differences in protein accumulation have greatly improved over the last decade and have shifted from gel image-based quantification to quantification within the mass spectrometer (37–39). Taking advantage of these new developments and opportunities, we compared the leaf proteome of clpr2-1 and wt seedlings early in development using spectral counting. This was complemented with a comparative analysis of the chloroplast soluble proteome of fully developed leaf rosettes. The seedling proteome analysis showed that the strongest effects occurred within the chloroplast. The functional significance of one of the most up-regulated proteins, ClpB3, was confirmed by additional mutant analysis. Putative substrates for the Clp system suggested in recent studies (28–30, 35) are reviewed in the context of our findings. This study provides testable hypotheses to further determine the significance of Clp-driven proteolysis and provides new insights in the plastid protein homeostasis network and how secondary metabolism is intertwined with photosynthetic capacity. We show that a systems view of chloroplast biogenesis and proteome homeostasis is needed to identify putative protease substrates and to understand the role of proteolysis in chloroplast biology. Finally we believe that the experimental setup described in this study provides an attractive template for comparative proteome analysis of other (chloroplast) mutants.     

Gene Coexpression Analysis Reveals Complex Metabolism of the Monoterpene Alcohol Linalool in Arabidopsis Flowers

The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpene synthases (TPS10 and TPS14) and the two cytochrome P450s (CYP71B31 and CYP76C3), are simultaneously expressed at anthesis, mainly in upper anther filaments and in petals. Upon transient expression in Nicotiana benthamiana, the TPS enzymes colocalize in vesicular structures associated with the plastid surface, whereas the P450 proteins were detected in the endoplasmic reticulum. Whether they were expressed in Saccharomyces cerevisiae or in N. benthamiana, the TPS enzymes formed two different enantiomers of linalool: (−)-(R)-linalool for TPS10 and (+)-(S)-linalool for TPS14. Both P450 enzymes metabolize the two linalool enantiomers to form different but overlapping sets of hydroxylated or epoxidized products. These oxygenated products are not emitted into the floral headspace, but accumulate in floral tissues as further converted or conjugated metabolites. This work reveals complex linalool metabolism in Arabidopsis flowers, the ecological role of which remains to be determined.