Plants possess two types of phosphofructokinase proteins for phosphorylation of fructose-6-phosph... more Plants possess two types of phosphofructokinase proteins for phosphorylation of fructose-6-phosphate, the ATP-dependent phosphofructokinase (PFK) and the pyrophosphate-(PPi) dependent pyrophosphate-fructose-6-phosphate-phosphotransferase (PFP). During oxygen deficiency ATP levels in rice seedlings are severely reduced, and it is hypothesized that PPi is used as an alternative energy source for the phosphorylation of fructose-6-phosphate during glycolysis. In this study, we analyzed the expression of 15 phosphofructokinaseencoding genes in roots and aerial tissues of anoxia-tolerant rice seedlings in response to anoxic stress and compared our data with transcript profiles obtained from microarray analyses. Furthermore, the intracellular localization of rice PFK proteins was determined, and the PFK and PFP isoforms were grouped in a phylogenetic tree. Two PFK and two PFP transcripts accumulated during anoxic stress, whereas mRNA levels of four PFK and three PFP genes were decreased. The total specific activity of both PFK and PFP changed only slightly during a 24-h anoxia treatment. It is assumed that expression of different isoforms and their catalytic properties differ during normoxic and anoxic conditions and contribute to balanced glycolytic activity during the low-oxygen stress. These characterizations of phosphofructokinase genes and the comparison to other plant species allowed us to suggest candidate rice genes for adaptation to anoxic stress.
The first committed and highly regulated step of chlorophyll biosynthesis is the insertion of Mg ... more The first committed and highly regulated step of chlorophyll biosynthesis is the insertion of Mg 2þ into protoporphyrin IX, which is catalyzed by Mg chelatase that consists of CHLH, CHLD and CHLI subunits. In this study, CHLI and CHLD genes were suppressed by virus-induced gene silencing (VIGS-CHLI and VIGS-CHLD) in pea (Pisum sativum), respectively. VIGS-CHLI and VIGS-CHLD plants both showed yellow leaf phenotypes with the reduced Mg chelatase activity and the inactivated synthesis of 5-aminolevulinic acid. The lower chlorophyll accumulation correlated with undeveloped thylakoid membranes, altered chloroplast nucleoid structure, malformed antenna complexes and compromised photosynthesis capacity in the yellow leaf tissues of the VIGS-CHLI and VIGS-CHLD plants. Non-enzymatic antioxidant contents and the activities of antioxidant enzymes were altered in response to enhanced accumulation of reactive oxygen species (ROS) in the chlorophyll deficient leaves of VIGS-CHLI and VIGS-CHLD plants. Furthermore, the results of metabolite profiling indicate a tight correlation between primary metabolic pathways and Mg chelatase activity. We also found that CHLD induces a feedback-regulated change of the transcription of photosynthesis-associated nuclear genes. CHLD and CHLI silencing resulted in a rapid reduction of photosynthetic proteins. Taken together, Mg chelatase is not only a key regulator of tetrapyrrole biosynthesis but its activity also correlates with ROS homeostasis, primary interorganellar metabolism and retrograde signaling in plant cells.
The levels of the mRNAs for light-inducible, nuclear-coded chloroplast proteins vary rhythmically... more The levels of the mRNAs for light-inducible, nuclear-coded chloroplast proteins vary rhythmically in pea (Pisum sativum L.) plants either grown in a dark-light cycle or under constant light conditions. This has been observed for the early light-inducible protein, the light-harvesting chlo- rophyll a/b protein, and the small subunit of the ribulose-1,5-bisphosphate carboxylase. The mRNA levels are high in the morning, exhibit a minimum in the first half of the night, and increase again during the second half of the night. The amplitude of fluctuation is between 5-and 10-fold. A similar change in the mRNA abundance was found for four nuclear encoded heat-shock proteins of 18, 24, 26, and 30 kilodaltons. The ability of plants to transcribe heat-shock genes upon heat-shock for 2 hours varies through the day. The maxima for induction are found in the second half of the night and the morning. The minima are reached during the afternoon. The degree of fluctuation is between 3and 5-fold. The levels of mRNAs for cytosolic as well as for plastid heat-shock proteins oscillate in parallel. 'The authors are grateful for the generous financial support by the
Biotechnologia. Journal of Biotechnology, Computational Biology and Bionanotechnology, 2013
Eukaryotic genes typically contain introns that are removed post-transcriptionally from the precu... more Eukaryotic genes typically contain introns that are removed post-transcriptionally from the precursor mRNA (pre-mRNA) through splicing. The presence of numerous exons per gene enables the splicing machinery to process the same pre-mRNA differently by selectively joining different exons, generating different transcripts from a single gene via a process named alternative splicing. In contrast to transcriptional control, alternative splicing can influence almost all aspects of protein function and has emerged as a key mechanism for generating proteome diversity and functional complexity. Its prevalence in many genomes, including those of higher plants, suggests that alternative splicing plays crucial roles in biological processes, as is emphasized by the fact that its misregulation can lead to many human diseases. However, information on the functional significance of this posttranscriptional regulation mechanism in plant systems is surprisingly scarce. We have identified an Arabidopsis thaliana gene, ZIFL1 , encoding a membrane transporter from the Major Facilitator Superfamily that plays important roles in both root auxin transport and drought stress tolerance. Selection of an alternative 3N splice site in the ZIFL1 pre-mRNA generates two splice variants that differ in only two nucleotides. While the longer transcript encodes the full-length transporter, the shorter contains a premature stop codon and codes for a truncated protein lacking the 67 C-terminal amino acids. Sequencing, promoter-reporter gene and fluorescent protein fusion experiments indicate that the full-length protein localizes specifically at the tonoplast of root cells, whereas the C-terminal truncation targets the transporter to the plasma membrane of stomatal guard cells. Using reverse genetics, we show that the root tonoplast-localized transporter regulates various auxin-related processes, while the truncated protein mediates drought tolerance by regulating stomatal closure. Heterologous expression in yeast revealed that the two splice forms share proton-coupled potassium transport activity. Thus, by determining the subcellular and tissue localization of two isoforms, alternative splicing allows the same gene to fulfill two very different but equally relevant roles in the plant.
Ludwig-Maximilians-Universität München eBooks, 2006
Supplement to chapter 1 An Overview of Chlorophylls and Bacteriochlorophylls: Biochemistry, Bioph... more Supplement to chapter 1 An Overview of Chlorophylls and Bacteriochlorophylls: Biochemistry, Biophysics, Functions and Applications (H. Scheer) All formulas in chapter 1 have been generated with the freeware program "C-Design" (J. Bauer, E. Fontain), which is available under: Sample formulas and schemes prepared with "C-Design" (ending .cdw) can be downloaded from this page. They require C-Design for opening and editing. A non-editable compilation in AdobeWriter format (.pdf) can be downloaded here. Major chlorophylls, with phytol and farnesol Chlorophylls a and b (IUPAC numbered) Chlorin-type chlorophylls and several esterifying alcohols Bacteriochlorophyll a (IUPAC numbered) Metallo bacteriochlorophyllls Protochlorophyllide a Chlorophyll enols (partial structures) Chlorophyll Biosynthesis: Overview From protoporphyrin IX to phlorophylls Porphobilinogen cyclization to protoporphyrin Chlorophyll nomenclature Fischer (left) vs. IUPAC numbering (right) Numbering, ß-ligand, and spectroscopic axes Others Major tetrapyrrole formulas Z-scheme of photosynthesis
The reactions occurring when glutamate-1-semialdehyde amino- transferase (glutamate-i -semialdehy... more The reactions occurring when glutamate-1-semialdehyde amino- transferase (glutamate-i -semialdehyde 2,1 aminomutase, EC 5.4.3.8) was treated with two potential mechanism-based inacti- vators, namely 4-aminohex-5-enoate and 4-aminohex-5-ynoate, have been investigated by monitoring rapid transient changes in the absorption spectrum of the enzyme's prosthetic group, pyridoxal 5'-phosphate. In both cases a short-lived chromophore absorbing maximally at about 500 rum was formed in a few milliseconds. In the case of the vinyl analogue (4-aminohex-5- enoate) this chromophore, considered to be a quinonoid in- termediate, converted rapidly into the pyridoxamine phosphate form of the co-enzyme in a single turnover which was accompanied by negligible inactivation. However, slow inactivation
Glutamate-l-semialdehyde aminotransferase (E.C. 5.4.3.8) was purified from barley and the cyanoba... more Glutamate-l-semialdehyde aminotransferase (E.C. 5.4.3.8) was purified from barley and the cyanobacteria Synechococcus PCC 6301. The purification procedure involved serial affinity chromatography and preparative polyacrylamide gel electrophoresis under non-denaturing conditions. The aminotransferase of these two organisms showed different mobilities in non-denaturing gels. In SDS-PAGE the enzyme from both organisms migrated as a single protein with an apparent molecular weight of 46.000 Da. An antibody against the barley enzyme cross-reacted with the cyanobacterial aminotransferase. This antibody also recognized a 17 kDa peptide cleaved from the barley protein with cyanogen bromide. Amino acid sequences of the NH2-termini revealed significant homology between the eucaryotic and cyanobacterial enzyme.
Arabidopsis HEMA1 and HEMA2 encode glutamyl-tRNA reductase (GluTR) 1 and 2, the two isoforms of t... more Arabidopsis HEMA1 and HEMA2 encode glutamyl-tRNA reductase (GluTR) 1 and 2, the two isoforms of the initial enzyme of tetrapyrrole biosynthesis. HEMA1 is dominantly expressed in photosynthetic tissue, while HEMA2 shows low constitutive expression and is induced upon stress treatments. We introduce a new HEMA1 knockout mutant which grows only heterotrophically on MS (Murashige and Skoog) medium at low light, indicating that the remaining GluTR2 does not sufficiently compensate for the extensive needs of metabolic precursors for Chl. While hema1 accumulates low amounts of Chl, it contains more than half of the wild-type heme content. The functional diversity of the two GluTR isoforms was analyzed by means of complementation studies of the hema1 mutant by expression of pHEMA1::HEMA2 and p35S::HEMA1, respectively. Expression of both transgenes complements hema1, indicating that GluTR2 can likewise be involved in the synthesis of Chl and is not exclusively assigned to heme synthesis. In comparison with p35S::HEMA1-complemented hema1 and the wild type, GluTR2 expression under control of the HEMA1 promoter (pHEMA1) in pHEMA1::HEMA2-complemented hema1 mutants causes elevated protochlorophyllide levels under extended dark periods as well as in shortday-grown adult plants, resulting in the formation of necrotic leaf tissue. Although both GluTR isoforms have similar activity and contribute to 5-aminolevulinic acid synthesis for adequate accumulation of Chl and heme, it is proposed that the two proteins experience a different post-translational control in darkness and light. While GluTR2 continues 5-aminolevulinic acid synthesis in darkness, GluTR1 is efficiently inactivated by the interaction with the FLU (FLUORESCENT) protein, thereby preventing an accumulation of protochlorophyllide.
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