title: Elevation of free proline and proline-rich protein levels by simultaneous manipulations of proline biosynthesis and degradation in plants.
abstract: Proline-rich proteins (PRP) are cell wall and plasma membrane-anchored factors involved in cell wall maintenance and its stress-induced fortification. Here we compare the synthesis of P5C as the proline (Pro) precursor in the cytosol and chloroplast by an introduced alien system and evaluate correlation between PRP synthesis and free Pro accumulation in plants. We developed a Pro over-producing system by generating transgenic tobacco plants overexpressing E. coli P5C biosynthetic enzymes; Pro-indifferent gamma-glutamyl kinase 74 (GK74) and gamma-glutamylphosphate reductase (GPR), as well as antisensing proline dehydrogenase (ProDH) transcription. GK74 and GPR enzymes were targeted either to the cytosol or plastids. Molecular analyses indicated that the two bacterial enzymes are efficiently expressed in plant cells, correctly targeted to the cytosol or chloroplasts, and processed to active enzymatic complexes in the two compartments. Maximal Pro increase is obtained when GK74 and GPR are active in chloroplasts, and ProDH mRNA level is reduced by anti-sense silencing, resulting in more than 50-fold higher Pro content compared to that of wild type tobacco plants. The Pro over-producing system efficiently works in tobacco and Arabidopsis. The elevation of Pro levels promotes accumulation of ectopically expressed Cell Wall Linker Protein (AtCWLP), a membrane protein with an external Pro-rich domain. These results suggest that the Pro-generating system can support endogenous or alien PRP production in plants.
authors.affiliation: Department of Plant Sciences, Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. hstein@cbd-tech.com
authors.forename: Hanan
authors.initials: H
authors.lastname: Stein
authors.forename: Arik
authors.initials: A
authors.lastname: Honig
authors.forename: Gad
authors.initials: G
authors.lastname: Miller
authors.forename: Oran
authors.initials: O
authors.lastname: Erster
authors.forename: Haviva
authors.initials: H
authors.lastname: Eilenberg
authors.forename: Laszlo N
authors.initials: LN
authors.lastname: Csonka
authors.forename: László
authors.initials: L
authors.lastname: Szabados
authors.forename: Csaba
authors.initials: C
authors.lastname: Koncz
authors.forename: Aviah
authors.initials: A
authors.lastname: Zilberstein
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D050698:Glutamate-5-Semialdehyde Dehydrogenase / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D000455:Medicago sativa / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D011392:Proline / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D011394:Proline Oxidase / Q000235:genetics / Q000378:metabolism; D011758:Pyrroles / Q000378:metabolism; D016372:RNA, Antisense / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D054712:Salinity; D013312:Stress, Physiological; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D010940:Plant Proteins; D011758:Pyrroles; D016372:RNA, Antisense; D018749:RNA, Plant; C015485:delta-1-pyrroline-5-carboxylate; D011392:Proline; D050698:Glutamate-5-Semialdehyde Dehydrogenase; D011394:Proline Oxidase; D017851:Phosphotransferases (Carboxyl Group Acceptor); C020650:glutamate 5-kinase