Arabidopsis ETHYLENE INSENSITIVE 3 directly regulates the expression of PG1ß-like family genes in response to aluminum stress.

The genes in the subfamily PG1ß (beta subunit of poly-galacturonase isoenzyme 1) have a clear effect on the biosynthesis pathway of pectin, a main component of the cell wall. However, the detailed functions of the PG1ß-like gene members in Arabidopsis (AtPG1-3) have not yet been determined. In this study, we investigated their functional roles in response to aluminum (Al) stress. Our results indicate that the PG1ß-like gene members are indeed involved in the Al-stress response and they can modulate its accumulation in roots to achieve optimum root elongation and hence better seedling growth. We found that transcription factor EIN3 (ETHYLENE INSENSITIVE 3) alters pectin metabolism and the EIN3 gene responds to Al stress to affect the pectin content in the root cell walls, leading to exacerbation of the inhibition of root growth, as reflected by the phenotypes of overexpressing lines. We determined that EIN3 can directly bind to the promoter regions of PG1-3, which act downstream of EIN3. Thus, our results show that EIN3 responds to Al stress in Arabidopsis directly through regulating the expression of PG1-3. Hence, EIN3 mediates their functions by acting as a biomarker in their molecular biosynthesis pathways, and consequently orchestrates their biological network in response to Al stress.

Ethylene insensitive3-like2 (OsEIL2) confers stress sensitivity by regulating OsBURP16, the ß subunit of polygalacturonase (PG1ß-like) subfamily gene in rice.

The transcription factors EIN3 (ETHYLENE-INSENSITIVE 3) and EILs (EIN3-Likes) play important roles in plant development and defense responses; however, their mechanism in these processes remain unclear. Here, we report that OsEIL2, an EIN3-like transcription factor from rice (Oryza sativa), plays important roles in abiotic stress and leaf senescence. OsEIL2 is a nuclear-localized protein with transactivation activity in the C-terminus (amino acids 344-583) and can be induced by NaCl, polyethylene glycol (PEG), dark, and abscisic acid (ABA) treatment. Transgenic plants of overexpressing OsEIL2 (OsEIL2-OX) show reduced tolerance to salt and drought stress compared with the controls. While the transgenic plants of overexpressing OsEIL2-RNA interference (OsEIL2-RNAi) exhibit enhanced tolerance to salt and drought stress compared with the controls. Moreover, seedlings of OsEIL2-overexpressing transgenic plants exhibit delayed leaf development and an accelerated dark-induced senescence phenotype, whereas OsEIL2-RNAi plants display the opposite phenotype. We further found that OsEIL2 functions upstream of OsBURP14 and OsBURP16. OsBURP14 and OsBURP16 are the members of the ß subunit of polygalacturonase subfamilies. OsBURP16 overexpression reduced pectin content and cell adhesion and increased abiotic stress sensitivity in rice. OsEIL2 binds directly to the promoter of OsBURP14 and OsBURP16 and activates their transcript levels. We also found that OsEIL2 overexpression decreased the pectin content by increasing polygalacturonase (PG) activity. Taken together, these results revealed a new mechanism of OsEIL2 in abiotic stress responses. These findings provide new insights into plant resistance to abiotic stress.

An in vitro DNA phosphorothioate modification reaction.

Phosphorothioation (PT) involves the replacement of a nonbridging phosphate oxygen on the DNA backbone with sulfur. In bacteria, the procedure is both sequence- and stereo-specific. We reconstituted the PT reaction using purified DndCDE from Salmonella enterica and IscS from Escherichia coli. We determined that the in vitro process of PT was oxygen sensitive. Only one strand on a double-stranded (ds) DNA substrate was modified in the reaction. The modification was dominant between G and A in the GAAC/GTTC conserved sequence. The modification between G and T required the presence of PT between G and A on the opposite strand. Cysteine, S-adenosyl methionine (SAM) and the formation of an iron-sulfur cluster in DndCDE (DndCDE-FeS) were essential for the process. Results from SAM cleavage reactions support the supposition that PT is a radical SAM reaction. Adenosine triphosphate (ATP) promoted the reaction but was not essential. The data and conclusions presented suggest that the PT reaction in bacteria involves three steps. The first step is the binding of DndCDE-FeS to DNA and searching for the modification sequence, possibly with the help of ATP. Cysteine locks DndCDE-FeS to the modification site with an appropriate protein conformation. SAM triggers the radical SAM reaction to complete the oxygen-sulfur swapping.

Phosphorothioated DNA Is Shielded from Oxidative Damage.

DNA is the carrier of genetic information. DNA modifications play a central role in essential physiological processes. Phosphorothioation (PT) modification involves the replacement of an oxygen atom on the DNA backbone with a sulfur atom. PT modification can cause genomic instability in Salmonella enterica under hypochlorous acid stress. This modification restores hydrogen peroxide (H2O2) resistance in the catalase-deficient Escherichia coli Hpx- strain. Here, we report biochemical characterization results for a purified PT modification protein complex (DndCDE) from S. enterica We observed multiplex oligomeric states of DndCDE by using native PAGE. This protein complex bound avidly to PT-modified DNA. DndCDE with an intact iron-sulfur cluster (DndCDE-FeS) possessed H2O2 decomposition activity, with a Vmax of 10.58 ± 0.90 mM min-1 and a half-saturation constant, K0.5S, of 31.03 mM. The Hill coefficient was 2.419 ± 0.59 for this activity. The protein's activity toward H2O2 was observed to be dependent on the intact DndCDE and on the formation of an iron-sulfur (Fe-S) cluster on the DndC subunit. In addition to cysteine residues that mediate the formation of this Fe-S cluster, other cysteine residues play a catalytic role. Finally, catalase activity was also detected in DndCDE from Pseudomonas fluorescens Pf0-1. The data and conclusions presented suggest that DndCDE-FeS is a short-lived catalase. Our experiments also indicate that the complex binds to PT sites, shielding PT DNA from H2O2 damage. This catalase shield might be able to extend from PT sites to the entire bacterial genome.IMPORTANCE DNA phosphorothioation has been reported in many bacteria. These PT-hosting bacteria live in very different environments, such as the human body, soil, or hot springs. The physiological function of DNA PT modification is still elusive. A remarkable property of PT modification is that purified genomic PT DNA is susceptible to oxidative cleavage. Among the oxidants, hypochlorous acid and H2O2 are of physiological relevance for human pathogens since they are generated during the human inflammation response to bacterial infection. However, expression of PT genes in the catalase-deficient E. coli Hpx- strain restores H2O2 resistance. Here, we seek to solve this obvious paradox. We demonstrate that DndCDE-FeS is a short-lived catalase that binds tightly to PT DNA. It is thus possible that by docking to PT sites the catalase activity protects the bacterial genome against H2O2 damage.

Identification of Non-Electrophilic Nrf2 Activators from Approved Drugs.

Oxidative damage can lead to a wide range of diseases. Nrf2 is an important transcription factor that regulates many of the cytoprotective enzymes involved in the oxidative stress response. Therefore, targeting the regulation of Nrf2 activation is one logical and effective strategy to prevent or lower the risk of oxidative stress-related diseases. Until now, most research has focused on electrophilic indirect Nrf2 activators, but the risk of 'off-target' effects may be associated with these activators. To find novel small non-electrophilic modulators of Nrf2, we started from chemical agents derived from a connectivity map (cMap) and identified 22 non-electrophilic potential Nrf2-activating drugs through a drug repositioning tactic. By determining the expression changes of antioxidant genes in MCF7 cells that were treated with the potential Nrf2 activators using quantitative real-time polymerase chain reaction RT-PCR (real-time polymerase chain reaction) (qRT-PCR), astemizole was found to have a greater scale of upregulating antioxidant genes NQO1, HO-1, and GCLM than the positive control d,l-sulforaphane, although the testing concentration was lower than that of the control. Astemizole is a good potential redox regulator and deserves more pharmacodynamic experimentation to test and verify its feasibility for use as an Nrf2 activator.

OsGA2ox5, a gibberellin metabolism enzyme, is involved in plant growth, the root gravity response and salt stress.

Gibberellin (GA) 2-oxidases play an important role in the GA catabolic pathway through 2ß-hydroxylation. There are two classes of GA2oxs, i.e., a larger class of C19-GA2oxs and a smaller class of C20-GA2oxs. In this study, the gene encoding a GA 2-oxidase of rice, Oryza sativa GA 2-oxidase 5 (OsGA2ox5), was cloned and characterized. BLASTP analysis showed that OsGA2ox5 belongs to the C20-GA2oxs subfamily, a subfamily of GA2oxs acting on C20-GAs (GA12, GA53). Subcellular localization of OsGA2ox5-YFP in transiently transformed onion epidermal cells revealed the presence of this protein in both of the nucleus and cytoplasm. Real-time PCR analysis, along with GUS staining, revealed that OsGA2ox5 is expressed in the roots, culms, leaves, sheaths and panicles of rice. Rice plants overexpressing OsGA2ox5 exhibited dominant dwarf and GA-deficient phenotypes, with shorter stems and later development of reproductive organs than the wild type. The dwarfism phenotype was partially rescued by the application of exogenous GA3 at a concentration of 10 µM. Ectopic expression of OsGA2ox5 cDNA in Arabidopsis resulted in a similar phenotype. Real-time PCR assays revealed that both GA synthesis-related genes and GA signaling genes were expressed at higher levels in transgenic rice plants than in wild-type rice; OsGA3ox1, which encodes a key enzyme in the last step of the bioactive GAs synthesis pathway, was highly expressed in transgenic rice. The roots of OsGA2ox5-ox plants exhibited increased starch granule accumulation and gravity responses, revealing a role for GA in root starch granule development and gravity responses. Furthermore, rice and Arabidopsis plants overexpressing OsGA2ox5 were more resistant to high-salinity stress than wild-type plants. These results suggest that OsGA2ox5 plays important roles in GAs homeostasis, development, gravity responses and stress tolerance in rice.

Microarray analyses and comparisons of upper or lower flanks of rice shoot base preceding gravitropic bending.

Gravitropism is a complex process involving a series of physiological pathways. Despite ongoing research, gravitropism sensing and response mechanisms are not well understood. To identify the key transcripts and corresponding pathways in gravitropism, a whole-genome microarray approach was used to analyze transcript abundance in the shoot base of rice (Oryza sativa sp. japonica) at 0.5 h and 6 h after gravistimulation by horizontal reorientation. Between upper and lower flanks of the shoot base, 167 transcripts at 0.5 h and 1202 transcripts at 6 h were discovered to be significantly different in abundance by 2-fold. Among these transcripts, 48 were found to be changed both at 0.5 h and 6 h, while 119 transcripts were only changed at 0.5 h and 1154 transcripts were changed at 6 h in association with gravitropism. MapMan and PageMan analyses were used to identify transcripts significantly changed in abundance. The asymmetric regulation of transcripts related to phytohormones, signaling, RNA transcription, metabolism and cell wall-related categories between upper and lower flanks were demonstrated. Potential roles of the identified transcripts in gravitropism are discussed. Our results suggest that the induction of asymmetrical transcription, likely as a consequence of gravitropic reorientation, precedes gravitropic bending in the rice shoot base.