Identification of Maize CC-Type Glutaredoxins That Are Associated with Response to Drought Stress

Global maize cultivation is often adversely affected by drought stress. The CC-type glutaredoxin (GRX) genes form a plant-specific subfamily that regulate plant growth and respond to environmental stresses. However, how maize CC-type GRX (ZmGRXCC) genes respond to drought stress remains unclear. We performed a TBLASTN search to identify ZmGRXCCs in the maize genome and verified the identified sequences using the NCBI conservative domain database (CDD). We further established a phylogenetic tree using Mega7 and surveyed known cis-elements in the promoters of ZmGRXCCs using the PlantCARE database. We found twenty-one ZmGRXCCs in the maize genome by a genome-wide investigation and compared their phylogenetic relationships with rice, maize, and Arabidopsis. The analysis of their redox active sites showed that most of the 21 ZmGRXCCs share similar structures with their homologs. We assessed their expression at young seedlings and adult leaves under drought stress and their expression profiles in 15 tissues, and found that they were differentially expressed, indicating that different ZmGRXCC genes have different functions. Notably, ZmGRXCC14 is up-regulated at seedling, V12, V14, V16, and R1 stages. Importantly, significant associations between genetic variation in ZmGRXCC14 and drought tolerance are found at the seedling stage. These results will help to advance the study of the function of ZmGRXCCs genes under drought stress and understand the mechanism of drought resistance in maize.

S-Glutathionyl-(chloro)hydroquinone reductases: a novel class of glutathione transferases

Sphingobium chlorophenolicum completely mineralizes PCP (pentachlorophenol). Two GSTs (glutathione transferases), PcpC and PcpF, are involved in the degradation. PcpC uses GSH to reduce TeCH (tetrachloro-p-hydroquinone) to TriCH (trichloro-p-hydroquinone) and then to DiCH (dichloro-p-hydroquinone) during PCP degradation. However, oxidatively damaged PcpC produces GS-TriCH (S-glutathionyl-TriCH) and GS-DiCH (S-glutathionyl-TriCH) conjugates. PcpF converts the conjugates into TriCH and DiCH, re-entering the degradation pathway. PcpF was further characterized in the present study. It catalysed GSH-dependent reduction of GS-TriCH via a Ping Pong mechanism. First, PcpF reacted with GS-TriCH to release TriCH and formed disulfide bond between its Cys53 residue and the GS moiety. Then, a GSH came in to regenerate PcpF and release GS–SG. A TBLASTN search revealed that PcpF homologues were widely distributed in bacteria, halobacteria (archaea), fungi and plants, and they belonged to ECM4 (extracellular mutant 4) group COG0435 in the conserved domain database. Phylogenetic analysis grouped PcpF and homologues into a distinct group, separated from Omega class GSTs. The two groups shared conserved amino acid residues, for GSH binding, but had different residues for the binding of the second substrate. Several recombinant PcpF homologues and two human Omega class GSTs were produced in Escherichia coli and purified. They had zero or low activities for transferring GSH to standard substrates, but all had reasonable activities for GSH-dependent reduction of disulfide bond (thiol transfer), dehydroascorbate and dimethylarsinate. All the tested PcpF homologues reduced GS-TriCH, but the two Omega class GSTs did not. Thus PcpF homologues were tentatively named S-glutathionyl-(chloro)hydroquinone reductases for catalysing the GSH-dependent reduction of GS-TriCH.

A Green Nonsulfur Bacterium, Dehalococcoides ethenogenes, with the LexA Binding Sequence Found in Gram-Positive Organisms

ABSTRACT Dehalococcoides ethenogenes is a member of the physiologically diverse division of green nonsulfur bacteria. Using a TBLASTN search, the D. ethenogenes lexA gene has been identified, cloned, and expressed and its protein has been purified. Mobility shift assays revealed that the D. ethenogenes LexA protein specifically binds to both its own promoter and that of the uvrA gene, but not to the recA promoter. Our results demonstrate that the D. ethenogenes LexA binding site is GAACN4GTTC, which is identical to that found in gram-positive bacteria. In agreement with this fact, the Bacillus subtilis DinR protein binds specifically to the D. ethenogenes LexA operator. This constitutes the first non-gram-positive bacterium exhibiting a LexA binding site identical to that of B. subtilis.

Cloning and expression of a novel UDP-GlcNAc:α-d-mannoside β1,2-N-acetylglucosaminyltransferase homologous to UDP-GlcNAc:α-3-d-mannoside β1,2-N-acetylglucosaminyltransferase I

A TBLASTN search with human UDP-GlcNAc:α-3-d-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101) as a probe identified human and mouse Unigenes encoding a protein similar to human GnT I (34% identity over 340 amino acids). The recombinant protein converted Man(α1–6)[Man(α1–3)]Man(β1-)O-octyl to Man(α1–6)[GlcNAc(β1–2)Man(α1–3)]Man(β1-)O-octyl, the reaction catalysed by GnT I. The enzyme also added GlcNAc to Man(α1–6)[GlcNAc(β1–2)Man(α1–3)]Man(β1-)O-octyl (the substrate for β-1,2-N-acetylglucosaminyltransferase II), Man(α1-)O-benzyl [with Km values of ≈ 0.3 and > 30mM for UDP-GlcNAc and Man(α1-)O-benzyl respectively] and the glycopeptide CYA[Man(α1-)O-T]AV (Km ∼ 12mM). The product formed with Man(α1-)O-benzyl was identified as GlcNAc(β1–2)Man(α1-)O-benzyl by proton NMR spectroscopy. The enzyme was named UDP-GlcNAc:α-d-mannoside β-1,2-N-acetylglucosaminyltransferase I.2 (GnT I.2). The human gene mapped to chromosome 1. Northern-blot analysis showed a 3.3kb message with a wide tissue distribution. The cDNA has a 1980bp open reading frame encoding a 660 amino acid protein with a type-2 domain structure typical of glycosyltransferases. Man(β1-)O-octyl, Man(β1-)O-p-nitrophenyl and GlcNAc(β1–2)Man(α1–6)[GlcNAc(β1–2)Man(α1–3)]Man(β1–4)GlcNAc(β1–4)GlcNAc(β1-)O-Asn were not acceptors, indicating that GnT I.2 is specific for α-linked terminal Man and does not have N-acetylglucosaminyltransferase III, IV, V, VII or VIII activities. CYA[Man(α1-)O-T]AV was between three and seven times more effective as an acceptor than the other substrates, suggesting that GnT I.2 may be responsible for the synthesis of the GlcNAc(β1–2)Man(α1-)O-Ser/Thr moiety on α-dystroglycan and other O-mannosylated proteins.