Comprehensive Derivatization of Thioviridamides by Heterologous Expression.

New technology for the derivatization of peptide natural products is required for drug development. Despite the recent advances in the genome sequencing technique enabling us to search for the biosynthetic genes for wide variety of natural products, the technical methods to get access to them are limited. A class of RiPPs, a recently emerged natural product family such as thioviridamide, is one of those possessing such unexplored chemical space. In this paper, we report a streamlined method to generate new thioviridamide derivatives and to assess their biological activities. Heterologous expression of 42 constructs in an engineered Streptomyces avermitilis host gave 35 designed thioviridamide derivatives, along with several unprecedented analogues. Moreover, cytotoxicity assay revealed that several derivatives showed more potent activities than those of prethioviridamide. These results indicate that this strategy can become one of the potential ways to produce supreme unnatural products.

Neothioviridamide, a Polythioamide Compound Produced by Heterologous Expression of a Streptomyces sp. Cryptic RiPP Biosynthetic Gene Cluster.

During genome mining for thioviridamide-like biosynthetic gene clusters that could produce polythioamide RiPP (ribosomally synthesized and post-translationally modified peptides), we discovered a novel cryptic biosynthetic gene cluster. During efforts to express this biosynthetic gene using heterologous expression of this biosynthetic gene cluster, a novel compound designated as neothioviridamide was produced. We report herein the cloning and heterologous expression of the neothioviridamide biosynthetic gene cluster and the isolation, structure determination, and cytotoxic activity of neothioviridamide.

Rice ubiquitin ligase EL5 prevents root meristematic cell death under high nitrogen conditions and interacts with a cytosolic GAPDH.

Root formation in rice transformants overexpressing mutated EL5 (mEL5) was severely inhibited because of meristematic cell death. Cell death was caused by nitrogen sources, particularly nitrate forms, in the culture medium. Nitrite treatment increased the cytokinin contents in roots, but mEL5 contained more cytokinins than non-transformants. Transcriptome profiling showed overlaps between nitrite-responsive genes in non-transformants and genes with altered expression in untreated mEL5. These results indicate that impairment of EL5 function activates nitrogen signaling despite the absence of a nitrogen source. Physical interaction between the EL5 C-terminal region and a cytosolic glyceraldehyde-3-phosphate dehydrogenase, OsGapC2, was demonstrated in vitro and in vivo. Elucidation of the role of glyceraldehyde-3-phosphate dehydrogenase in oxidative cell death in plants is expected in future.

Ubiquitin ligase EL5 maintains the viability of root meristems by influencing cytokinin-mediated nitrogen effects in rice.

Root formation is dependent on meristematic activity and is influenced by nitrogen supply. We have previously shown that ubiquitin ligase, EL5, in rice (Oryza sativa) is involved in the maintenance of root meristematic viability. When mutant EL5 protein is overexpressed to dominantly inhibit the endogenous EL5 function in rice, primordial and meristematic necrosis ia observed. Here, we analysed the cause of root cell death in transgenic rice plants (mEL5) overexpressing EL5V162A, which encodes a partly inactive ubiquitin ligase. The mEL5 mutants showed increased sensitivity to nitrogen that was reflected in the inhibition of root formation. Treatment of mEL5 with nitrate or nitrite caused meristematic cell death accompanied by browning. Transcriptome profiling of whole roots exhibited overlaps between nitrite-responsive genes in non-transgenic (NT) rice plants and genes with altered basal expression levels in mEL5. Phytohormone profiling of whole roots revealed that nitrite treatment increased cytokinin levels, but mEL5 constitutively contained more cytokinin than NT plants and showed increased sensitivity to exogenous cytokinin. More superoxide was detected in mEL5 roots after treatment with nitrite or cytokinin, and treatment with an inhibitor of superoxide production prevented mEL5 roots from both nitrite- and cytokinin-induced meristematic cell death. These results indicate a nitrogen-triggered pathway that leads to changes in root formation through the production of cytokinin and superoxide, on which EL5 acts to prevent meristematic cell death.

Engineered Streptomyces avermitilis host for heterologous expression of biosynthetic gene cluster for secondary metabolites.

An industrial microorganism, Streptomyces avermitilis, which is a producer of anthelmintic macrocyclic lactones, avermectins, has been constructed as a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis. Twenty of the entire biosynthetic gene clusters for secondary metabolites were successively cloned and introduced into a versatile model host S. avermitilis SUKA17 or 22. Almost all S. avermitilis transformants carrying the entire gene cluster produced metabolites as a result of the expression of biosynthetic gene clusters introduced. A few transformants were unable to produce metabolites, but their production was restored by the expression of biosynthetic genes using an alternative promoter or the expression of a regulatory gene in the gene cluster that controls the expression of biosynthetic genes in the cluster using an alternative promoter. Production of metabolites in some transformants of the versatile host was higher than that of the original producers, and cryptic biosynthetic gene clusters in the original producer were also expressed in a versatile host.

Drought induction of Arabidopsis 9-cis-epoxycarotenoid dioxygenase occurs in vascular parenchyma cells.

The regulation of abscisic acid (ABA) biosynthesis is essential for plant responses to drought stress. In this study, we examined the tissue-specific localization of ABA biosynthetic enzymes in turgid and dehydrated Arabidopsis (Arabidopsis thaliana) plants using specific antibodies against 9-cis-epoxycarotenoid dioxygenase 3 (AtNCED3), AtABA2, and Arabidopsis aldehyde oxidase 3 (AAO3). Immunohistochemical analysis revealed that in turgid plants, AtABA2 and AAO3 proteins were localized in vascular parenchyma cells most abundantly at the boundary between xylem and phloem bundles, but the AtNCED3 protein was undetectable in these tissues. In water-stressed plants, AtNCED3 was detected exclusively in the vascular parenchyma cells together with AtABA2 and AAO3. In situ hybridization using the antisense probe for AtNCED3 showed that the drought-induced expression of AtNCED3 was also restricted to the vascular tissues. Expression analysis of laser-microdissected cells revealed that, among nine drought-inducible genes examined, the early induction of most genes was spatially restricted to vascular cells at 1 h and then some spread to mesophyll cells at 3 h. The spatial constraint of AtNCED3 expression in vascular tissues provides a novel insight into plant systemic response to drought stresses.

EL5 is involved in root development as an anti-cell death ubiquitin ligase.

Ubiquitin ligase (E3) plays a central role in substrate recognition during ubiquitination, a post-translational modification of proteins. Rice EL5 is an E3 with a RING-H2 finger domain (RFD) and its transcript is upregulated by a chitin elicitor. The EL5-RFD has been intensively studied and demonstrated to exhibit E3 activity. Its three-dimensional structure was determined for the first time in plant E3, and the amino acid residues required for the interaction with the ubiquitin-conjugating enzyme (E2) were identified. Recent analyses revealed that EL5 plays a crucial role as an E3 in the maintenance of cell viability during root development in rice. In this addendum, we report that the EL5-RFD catalyzes polyubiquitination via the Lys48 residue of ubiquitin. We also discuss the possible role of EL5 as an anti-cell death enzyme. We hypothesize that EL5 might be responsible for mediating the degradation of cytotoxic proteins produced in root cells after the actions of phytohormones.

Transient expression of AtNCED3 and AAO3 genes in guard cells causes stomatal closure in Vicia faba.

Abscisic acid (ABA) regulates stomatal closure in response to water loss. Here, we examined the competence of guard cells to synthesize ABA, using two Arabidopsis ABA biosynthetic enzymes. 35S pro::AtNCED3-GFP and AAO3-GFP were introduced into guard cells of broad bean leaves. AtNCED3-GFP expression was detected at the chloroplasts, whereas green fluorescent protein (GFP) and AAO3-GFP were in the cytosol. The stomatal aperture was decreased in AtNCED3-GFP- and AAO3-GFP-transformed guard cells. This indicated that ABA biosynthesis is stimulated by heterologous expression of AtNCED3 and Arabidopsis aldehyde oxidase 3 (AAO3) proteins, which both seem to be regulatory enzymes for ABA biosynthesis in these cells. Furthermore, stomatal closure by the expression of AtNCED3 and AAO3 suggested that the substrates of the enzymes are present and native ABA-biosynthesis enzymes are active in guard cells.

RING-H2 type ubiquitin ligase EL5 is involved in root development through the maintenance of cell viability in rice.

Rice EL5 is an ATL family gene characterized by a transmembrane domain at the N-terminal and a RING-H2 finger domain (RFD), which exhibits ubiquitin ligase (E3) activity. To elucidate the physiological roles of EL5, we analyzed transgenic rice plants overexpressing mutant EL5 proteins that are impaired in E3 activity to various degrees. Plants expressing EL5C153A and EL5W165A, which encode an inactive E3, showed a rootless phenotype accompanied by cell death in root primordia, and those expressing EL5V162A, with moderately impaired E3 activity, formed short crown roots with necrotic lateral roots. The dominant-negative phenotype was specifically observed in root meristems where EL5 is expressed, and not recovered by exogenous auxin. When wild-type EL5 was transcriptionally overexpressed, the EL5 protein was barely detected by Western blotting. Neither treatment with a proteasome inhibitor nor mutation of the sole lysine residue, a potential target of ubiquitination, resulted in increased EL5 accumulation, whereas mutations in the RFD led to increased EL5 accumulation. The stabilized EL5 without the RFD was localized in the plasma membrane. Deletion of the transmembrane domain prevented the EL5 from localizing in the membrane and from exerting an inhibitory effect on root formation. Deletion of the C-terminal region also neutralized the negative effect. We concluded that EL5 plays a major role as a membrane-anchored E3 for the maintenance of cell viability after the initiation of root primordial formation. In addition, we propose that EL5 is an unstable protein, of which degradation is regulated by the RFD in a proteasome-independent manner.

Comparative studies on the Arabidopsis aldehyde oxidase (AAO) gene family revealed a major role of AAO3 in ABA biosynthesis in seeds.

The Arabidopsis aldehyde oxidase 3 (AAO3) gene encodes an enzyme that catalyzes the final step of ABA biosynthesis. AAO3 has been shown to be the major AAO involved in ABA biosynthesis in leaves under stress conditions. On the other hand, less severe phenotypes of the aao3 seeds suggested that other AAO(s) might also be involved in ABA biosynthesis in seeds. Among four AAOs (AAO1-AAO4), AAO1 and AAO4 were the AAO expressed most abundantly in dry seeds and developing siliques, respectively. Unlike aao3, single loss-of-function mutants for AAO1 and AAO4 (aao1 and aao4), failed to show significant changes in endogenous ABA levels in seeds when compared with wild type. While aao3 seed germination was resistant to the gibberellin biosynthesis inhibitor, uniconazole, aao1 and aao4 showed no resistance and were similar to wild type. These results indicate that AAO3, but not AAO1 or AAO4, plays an important role in ABA biosynthesis in seeds. Mutations of AAO1 or AAO4 in the aao3 mutant background enhanced ABA deficiency in seeds, demonstrating that both gene products contribute partially to ABA biosynthesis in the aao3 mutant background. However, considering the enzymatic characters of AAO1 and AAO4, their involvement in ABA biosynthesis in wild-type seeds may be negligible. We have concluded that AAO3 is the AAO that plays a major role in ABA biosynthesis in Arabidopsis seeds as well as in leaves.

Tissue-specific localization of an abscisic acid biosynthetic enzyme, AAO3, in Arabidopsis.

Arabidopsis aldehyde oxidase 3 (AAO3) is an enzyme involved in abscisic acid (ABA) biosynthesis in response to drought stress. Since the enzyme catalyzes the last step of the pathway, ABA production sites may be determined by the presence of AAO3. Here, AAO3 localization was investigated using AAO3 promoter:AAO3-GFP transgenic plants and by an immunohistochemical technique. AAO3-GFP protein exhibited an activity to produce ABA from abscisic aldehyde, and the transgene restored the wilty phenotype of the aao3 mutant. GFP-fluorescence was detected in the root tips, vascular bundles of roots, hypocotyls and inflorescence stems, and along the leaf veins. Intense immunofluorescence signals were localized in phloem companion cells and xylem parenchyma cells. Faint but significant GFP- and immuno-fluorescence signals were observed in the leaf guard cells. In situ hybridization with antisense AAO3 mRNA showed AAO3 mRNA expression in the guard cells of dehydrated leaves. These results indicate that the ABA synthesized in vascular systems is transported to various target tissues and cells, and also that the guard cells themselves are able to synthesize ABA.