Shinya Murakami

A rice fungal MAMP‐responsive MAPK cascade regulates metabolic flow to antimicrobial metabolite synthesis

Plants recognize potential microbial pathogens through microbial-associated molecular patterns (MAMPs) and activate a series of defense responses, including cell death and the production of reactive oxygen species (ROS) and diverse anti-microbial secondary metabolites. Mitogen-activated protein kinase (MAPK) cascades are known to play a pivotal role in mediating MAMP signals; however, the signaling pathway from a MAPK cascade to the activation of defense responses is poorly understood. Here, we found in rice that the chitin elicitor, a fungal MAMP, activates two rice MAPKs (OsMPK3 and OsMPK6) and one MAPK kinase (OsMKK4). OsMPK6 was essential for the chitin elicitor-induced biosynthesis of diterpenoid phytoalexins. Conditional expression of the active form of OsMKK4 (OsMKK4DD) induced extensive alterations in gene expression, which implied dynamic changes of metabolic flow from glycolysis to secondary metabolite biosynthesis while suppressing basic cellular activities such as translation and cell division. OsMKK4DD also induced various defense responses, such as cell death, biosynthesis of diterpenoid phytoalexins and lignin but not generation of extracellular ROS. OsMKK4DD-induced cell death and expression of diterpenoid phytoalexin pathway genes, but not that of phenylpropanoid pathway genes, were dependent on OsMPK6. Collectively, the OsMKK4–OsMPK6 cascade plays a crucial role in reprogramming plant metabolism during MAMP-triggered defense responses.

The DNA-binding protease, CND41, and the degradation of ribulose-1, 5-bisphosphate carboxylase/oxygenase in senescent leaves of tobacco

Plastids bear their own genome, organized into DNA–protein complexes (nucleoids). Recently, we identified a DNA-binding protease (CND41) in the chloroplast nucleoids of cultured tobacco (Nicotiana tabacum L.) cells. In this study, we examine the biochemical function of this novel DNA-binding protease, particularly in senescent leaves, because antisense tobacco with a reduced amount of CND41 showed retarded senescence. Nitrogen-depletion experiments clearly showed that CND41 antisense tobacco maintained green leaves and constant protein levels, especially ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), throughout the whole plant, whereas wild-type tobacco showed marked senescence and the reduction of protein levels in the lower leaves. In vitro analyses confirmed that CND41 showed proteolytic activity at physiological pH when denatured Rubisco was used as the substrate. These results suggest that CND41 is involved in Rubisco degradation and the translocation of nitrogen during senescence. The possible regulation of protease activity of CND41 through DNA-binding is discussed.

A novel protein with DNA binding activity from tobacco chloroplast nucleoids.

A 41-kD DNA binding protein with a basic pl was purified from chloroplast nucleoids in photomixotrophically cultured tobacco cells, and its amino acid sequence was determined. Using this sequence information, its cDNA (CND41) was isolated, and its nucleotide sequence was determined. The predicted amino acid sequence of CND41 has a transit peptide of 120 amino acids and a mature protein of 382 amino acids. A distinctive helix-turn-helix motif in the lysine-rich N-terminal region of the mature protein and an aspartyl protease active site motif were predicted. Expression of a series of truncated CND41 proteins in Escherichia coli indicated that the lysine-rich region is essential for DNA binding and that CND41 nonspecifically binds chloroplast DNA. Protein gel blot analyses showed CND41 mainly in cells and/or tissues containing nonphotosynthesizing, actively growing plastids. In addition, the accumulation of chloroplast transcripts in these cells and/or tissues (e.g., transcripts for QB binding protein of photosystem II [psbA] and large subunit of ribulose bisphosphate carboxylase [rbcL]) was negatively correlated with the accumulation of CND41. Analyses of cultured cells of transgenic tobacco with reduced CND41 levels showed a higher level of expression of chloroplast genes compared with that of the wild type. We discuss the possible function of CND41 as a negative regulator of chloroplast gene expression.

Protease activity of CND41, a chloroplast nucleoid DNA-binding protein, isolated from cultured tobacco cells.

CND41 is a 41 kDa DNA‐binding protein isolated from chloroplast nucleoids of cultured tobacco cells. The presence of the active domain of aspartic protease in the deduced amino acid sequence of CND41 suggests that it has proteolytic activity. To confirm this, CND41 was highly purified from cultured tobacco cells and its proteolytic activity was characterized with fluorescein isothiocyanate‐labeled hemoglobin as the substrate. The purified CND41 had strong proteolytic activity at an acidic pH (pH 2–4). This activity was inhibited by various chemicals, including the nucleoside triphosphates, NADPH, Fe3+ and sodium dodecyl sulfate.

Novel nuclear-encoded proteins interacting with a plastid sigma factor, Sig1, in Arabidopsis thaliana.

Sigma factor binding proteins are involved in modifying the promoter preferences of the RNA polymerase in bacteria. We found the nuclear encoded protein (SibI) that is transported into chloroplasts and interacts specifically with the region 4 of Sig1 in Arabidopsis. SibI and its homologue, T3K9.5 are novel proteins, which are not homologous to any protein of known function. The expression of sibI was tissue specific, light dependent, and developmentally timed. We suggest the transcriptional regulation by sigma factor binding proteins to function in the plastids of higher plant.

Regulation of CONIFERALDEHYDE 5 - HYDROXYLASE expression to modulate cell wall lignin structure in rice

AbstractMain conclusionRegulation of a gene encoding coniferaldehyde 5-hydroxylase leads to substantial alterations in lignin structure in rice cell walls, identifying a promising genetic engineering target for improving grass biomass utilization. The aromatic composition of lignin greatly affects utilization characteristics of lignocellulosic biomass and, therefore, has been one of the primary targets of cell wall engineering studies. Limited information is, however, available regarding lignin modifications in monocotyledonous grasses, despite the fact that grass lignocelluloses have a great potential for feedstocks of biofuel production and various biorefinery applications. Here, we report that manipulation of a gene encoding coniferaldehyde 5-hydroxylase (CAld5H, or ferulate 5-hydroxylase, F5H) leads to substantial alterations in syringyl (S)/guaiacyl (G) lignin aromatic composition in rice (Oryza sativa), a major model grass and commercially important crop. Among three CAld5H genes identified in rice, OsCAld5H1 (CYP84A5) appeared to be predominantly expressed in lignin-producing rice vegetative tissues. Down-regulation of OsCAld5H1 produced altered lignins largely enriched in G units, whereas up-regulation of OsCAld5H1 resulted in lignins enriched in S units, as revealed by a series of wet-chemical and NMR structural analyses. Our data collectively demonstrate that OsCAld5H1 expression is a major factor controlling S/G lignin composition in rice cell walls. Given that S/G lignin composition affects various biomass properties, we contemplate that manipulation of CAld5H gene expression represents a promising strategy to upgrade grass biomass for biorefinery applications.

Purification of CND41; Chloroplast Nucleoid Dna-Binding Protein with Proteolytic Activity

CND41 is the major DNA-binding protein of chloroplast nucleoid of photomixotrophically cultured tobacco cells (1). Its cDNA was isolated and DNA sequence was determined (2). The deduced amino acid sequence of CND41 has a transit peptide of 120 amino acids and a mature protein of 382 amino acids. Recombinant CND41 expressed in E. coil showed that lysine-rich region existing in N-terminal of mature protein was necessary for DNA binding. CND41 also has aspartic protease active site and considerable identity against mucoropepsin (about 25 %), an aspartic protease of Mucore miehei (3) over 380 amino acids, while the proteolytic activity has not been determined. The characterization of accumulation of CND41 indicated that there was a negative correlation between CND41 accumulation and chloroplast transcript levels. CND41 was abundant in non- or low-photosynthetic cells as stem and non-selected cultured cells, whereas CND41 level was low in photosynthetic cells like mature leaves and photoautotrophically cultured cells. Transgenic tobacco also indicated that tobacco with less CND41 had higher chloroplast transcripts. From these results, we proposed that CND41 might be a negative regulator for the chloroplast gene expression (2), while the function of DNA binding activity and/or protease activity of CND41 in the regulation was not clear yet. In this study, we purified CND41 and determined its proteolytic activity to characterize CND41 function in vivo.

Regulation of plastid gene transcription by sigma factor binding protein

In chloroplasts, many photosynthesis-related genes are transcribed tissue-specifically and/or light responsively by E.coli-type RNA polymerase (PEP). Core subunits of PEP are encoded by the plastid genome, while the promoter recognition is mediated by sigma factors that are encoded by the nuclear genome. The differential usage of the sigma factors is considered to play the pivotal role for the tissue-specific and/or light responsive transcription. Here, we identified a novel protein termed SibI that specifically binds to the region 4 (-35 element recognition domain) of a sigma factor (At Sig1) by yeast two-hybrid screening. SibI had no sequence similarity to any proteins of known function, but the N-terminal region of SibI had a feature of the chloroplast transit peptide. Transient expression assay of SibI-GFP fusion protein in protoplasts showed that SibI was imported into chloroplasts. We established a new run-on assay system utilizing transformed protoplasts, and found that the transient expression of SibI caused the gene specific transcriptional activation with various degrees depending on the protoplast preparations. These results suggest that the nuclear-encoded sigma factor binding protein, SibI, is involved in the nuclear-control over the plastid gene transcription. The molecular mechanism of the SibI function in the plastid gene transcription will be discussed.