Two Global Conformation States of a Novel NAD(P) Reductase Like Protein of the Thermogenic Appendix of the Sauromatum guttatum Inflorescence

A novel NAD(P) reductase like protein (RL) belonging to a class of reductases involved in phenylpropanoid synthesis was previously purified to homogeneity from the Sauromatum guttatum appendix. The Sauromatum appendix raises its temperature above ambient temperature to ~30 °C on the day of inflorescence opening (D-day). Changes in the charge state distribution of the protein in electrospray ionization–mass spectrometry spectra were observed during the development of the appendix. RL adopted two conformations, state A (an extended state) that appeared before heat-production (D ? 4 to D ? 2), and state B (a compact state) that began appearing on D ? 1 and reached a maximum on D-day. RL in healthy leaves of Arabidopsis is present in state A, whereas in thermogenic sporophylls of male cones of Encephalartos ferox is present in state B. These conformational changes strongly suggest an involvement of RL in heat-production. The biophysical properties of this protein are remarkable. It is self-assembled in aqueous solutions into micrometer sizes of organized morphologies. The assembly produces a broad range of cyclic and linear morphologies that resemble micelles, rods, lamellar micelles, as well as vesicles. The assemblies could also form network structures. RL molecules entangle with each other and formed branched, interconnected networks. These unusual assemblies suggest that RL is an oligomer, and its oligomerization can provide additional information needed for thermoregulation. We hypothesize that state A controls the plant basal temperature and state B allows a shift in the temperature set point to above ambient temperature.     

Human Ortholog of a Plant Salicylic Acid Receptor Found in SK-N-SH Cell Line

Our previous studies have described the purification and characterization of a novel plant NAD(P)-reductase like protein (RL) from the thermogenic appendix of the Sauromatum guttatum inflorescence. RL is mainly located in cytoplasm of thermogenic plants and it can act like a bistable switch. It adopts a compact conformation during heat-production and a more expanded conformation when heat is not generated. Addition of salicylic acid, a natural thermogenic inducer, at picomolar concentration to a solution of purified RL induced a discontinuous volume phase transition in which the volume of RL in the oligomeric form expanded and shrunk repeatedly every 4–5 min. In the present study using ESI–MS analysis we have demonstrated the existence of RL in the human SK-N-SH cell line and in mouse brain tissue. The molecular mass of human RL is in the same range as of its plant counterpart, 34,140 ± 34 Da. The charge state distribution of the human RL is identical to its plant counterpart from the Sauromatum appendix during heat-production. Human RL was present in the compact state when it was purified from the SK-N-SH cell line When these cells were treated with salicylic acid (10 μM) a shift to a much more compact conformation was observed. It seems that the potential of RL to respond to salicylic acid was conserved. These results may reveal the existence of a thermoregulation system that is evolutionarily conserved and is operating by conformational changes. This discovery may also represent an opportunity for a better understanding of some of the diverse functions of salicylic acid and aspirin in plants and humans.     

Oscillatory Heat-Production in the Male Cones of Cycads

Microcalorimetric analysis of heat-production by single intactsporophylls from thermogenic male cones of two cycad species(Ceratozamia miqueliana H. Wendl. and Zamia furfuracea L. f.)showed an oscillatory behaviour for three consecutive days duringpollen maturation and shedding. In sporophylls from C. rumphiiMiq., a weakly thermogenic species with no maximum of heat-production,such oscillations were not detected. The presence of the alternativerespiratory pathway in the sporophylls of the male cones ofZamia pumila L. has been demonstrated. Key words: Alternative pathway, cycads, heat-production, microcalorimetry, oscillation     

Energy Loss in Tissue 2Sauromatum guttatum (Schott) Analysed by Microcalorimetry

The heat evolved by 1 mm-thick tissue slices of the appendixof the Sauromatum guttatum inflorescence was measured calorimetricallyduring development. From D–5 (5 d before inflorescence-opening,designated as D-day) to D–2 about 8 µW mg^–1fresh wt. was observed, and during D–1 an increase inheat evolution to 14 µW mg^–1 fresh wt. was monitored.The heat was produced through oxidative metabolism, since replacingthe air in the microcalorimeter with nitrogen blocked heatproduction.Addition of salicylic acid to tissue slices of thermogenic organs(appendix, lowest part of the spadix and male flowers) and ofnon-thermogenic organs (female flowers, club-shaped organs andlower part of the spadix) of Sauromatum inflorescences revealedthat the acid boosted heat-production only in the thermogenicorgans. The effect of the acid manifested itself with no appreciablelag time, and it generated non-linearity in the rate of heat-productionby tissue slices of the appendix. In the appendices of the highlythermogenic Arum italicum and of the weakly thermogenic Amorphophallusrivieri, salicylic acid selectively boosted the rate of heat-productionin the appendix of A. italicum. A Q₁₀ of 24 was found between15C to 25 C for 1 mm-thick slices of D–4 appendicesof S guttatum. Addition of digitonin or deoxycholate to pre-D-daytissue slices of the appendix increased the rate of heat-production. Key words: Amorphophallus rivieri, Arum italicum, microcalorimetry, salicylic acid, Sauromatum guttatum     

Substrate flexibility and reaction specificity of tropinone reductase-like short-chain dehydrogenases

Annotations of protein or gene sequences from large scale sequencing projects are based on protein size, characteristic binding motifs, and conserved catalytic amino acids, but biochemical functions are often uncertain. In the large family of short-chain dehydrogenases/reductases (SDRs), functional predictions often fail. Putative tropinone reductases, named tropinone reductase-like (TRL), are SDRs annotated in many genomes of organisms that do not contain tropane alkaloids. SDRs in vitro often accept several substrates complicating functional assignments. Cochlearia officinalis, a Brassicaceae, contains tropane alkaloids, in contrast to the closely related Arabidopsis thaliana. TRLs from Arabidopsis and the tropinone reductase isolated from Cochlearia (CoTR) were investigated for their catalytic capacity. In contrast to CoTR, none of the Arabidopsis TRLs reduced tropinone in vitro. NAD(H) and NADP(H) preferences were relaxed in two TRLs, and protein homology models revealed flexibility of amino acid residues in the active site allowing binding of both cofactors. TRLs reduced various carbonyl compounds, among them terpene ketones. The reduction was stereospecific for most of TRLs investigated, and the corresponding terpene alcohol oxidation was stereoselective. Carbonyl compounds that were identified to serve as substrates were applied for modeling pharmacophores of each TRL. A database of commercially available compounds was screened using the pharmacophores. Compounds identified as potential substrates were confirmed by turnover in vitro. Thus pharmacophores may contribute to better predictability of biochemical functions of SDR enzymes.     

Heat Production in the Voodoo Lily (Sauromatum guttatum) as Monitored by Infrared Thermography

The pattern of surface temperatures of the inflorescence of Sauromatum guttatum was investigated by using an infrared camera. The male flowers are weakly thermogenic on the first day of inflorescence opening (D-day) as well as on the next day (D + 1), reaching 0.5 to 1°C above ambient temperature. The appendix (the upper sterile part of the inflorescence) is highly thermogenic on D-day, reaching 32°C, and is faintly thermogenic on D + 1, reaching 1°C above ambient temperature. The lower part of the spadix, close to the female flowers, is also thermogenic on D-day and D + 1, reaching a temperature similar to that of the appendix only on D + 1. Salicylic acid does not induce heat production in the lower part of the spadix, as it does in the appendix. Respiration of tissue slices obtained from the appendix shows that the capacity for cyanide-insensitive respiration is present in young and mature appendices. This alternative respiratory pathway is not, however, utilized in young appendix tissue, but is engaged during the maturation of that tissue.     

Overexpression of TaHSF3 in Transgenic Arabidopsis Enhances Tolerance to Extreme Temperatures

Heat shock factors (HSFs) in plants regulate heat stress response by mediating expression of a set of heat shock protein (HSP) genes. In the present study, we isolated a novel heat shock gene, TaHSF3, encoding a protein of 315 amino acids in wheat. Phylogenetic analysis showed that TaHSF3 belonged to HSF class B2. Subcellular localization analysis indicated that TaHSF3 localized in nuclei. TaHSF3 was highly expressed in wheat spikes and showed intermediate expression levels in roots, stems, and leaves under normal conditions. It was highly upregulated in wheat seedlings by heat and cold and to a lesser extent by drought and NaCl and ABA treatments. Overexpression of TaHSF3 in Arabidopsis enhanced tolerance to extreme temperatures. Frequency of survival of three TaHSF3 transgenic Arabidopsis lines was 75–91 % after heat treatment and 85–95 % after freezing treatment compared to 25 and 10 %, respectively, in wild-type plants (WT). Leaf chlorophyll contents of the transformants were higher (0.52–0.67 mg/g) than WT (0.35 mg/g) after heat treatment, and the relative electrical conductivities of the transformants after freezing treatment were lower (from 17.56 to 18.6 %) than those of WT (37.5 %). The TaHSF3 gene from wheat therefore confers tolerance to extreme temperatures in transgenic Arabidopsis by activating HSPs, such as HSP70.     

Anatomical distribution of primary amine oxidase activity in four adipose depots and plasma of severely obese women with or without a dysmetabolic profile

Semicarbazide-sensitive amine oxidase (SSAO), identical to primary amine oxidase or vascular adhesion protein-1, is a membrane enzyme that generates hydrogen peroxide. SSAO is highly expressed at the adipocyte surface, and its plasma levels increase with type 2 diabetes. Since visceral adipose tissue (AT) is more tightly associated with obesity complications than subcutaneous (SC) abdominal fat, we compared SSAO activity in plasma and 4 distinct AT locations in 48 severely obese women (body mass index (BMI), averaging 54 ± 11 kg/m²), with or without a dysmetabolic profile. Higher glucose and triacylglycerol levels vs lower high-density lipoprotein (HDL)-cholesterol characterized dysmetabolic women (DYS; n = 25) from non-dysmetabolic (NDYS; n = 23), age- and weight-matched subjects. SC, mesenteric (ME), omental (OM), and round ligament (RL) fat locations were collected during bariatric surgery. SSAO capacity to oxidize up to 1 mM benzylamine was determined in AT and plasma with radiometric and fluorimetric methods. Plasma SSAO was higher in the DYS group. SSAO activity was higher in fat than in plasma, when expressed as radiolabeled benzaldehyde per milligram of protein. In ATs from DYS women, protein content was 10 % higher, and basal hydrogen peroxide release lower than in NDYS subjects, except for RL location. The SSAO affinity towards benzylamine did not exhibit regional variation and was not altered by a dysmetabolic profile (K _m averaging 184 ± 7 μM; n = 183). Although radiometric and fluorimetric methods gave different estimates of oxidase activity, both indicated that AT SSAO activity did not vary according to anatomical location and/or metabolic status in severely obese women.     

Cloning, Large Scale Over-Expression in E. coli and Purification of the Components of the Human LAT 1 (SLC7A5) Amino Acid Transporter

The high yield expression of the human LAT1 transporter has been obtained for the first time using E. coli. The hLAT1 cDNA was amplified from HEK293 cells and cloned in pH6EX3 vector. The construct pH6EX3-6His-hLAT1 was used to express the 6His-hLAT1 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected 8 h after induction by IPTG at 28 °C. The expressed protein was collected in the insoluble fraction of cell lysate. On SDS-PAGE the apparent molecular mass of the polypeptide was 40 kDa. After solubilization with sarkosyl and denaturation with urea the protein carrying a 6His N-terminal tag was purified by Ni²⁺-chelating affinity chromatography and identified by anti-His antibody. The yield of the over-expressed protein after purification was 3.5 mg/L (cell culture). The human CD98 cDNA amplified from Imagene plasmid was cloned in pGEX-4T1. The construct pGEX-4T1-hCD98 was used to express the GST-hCD98 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected in this case 4 h after induction by IPTG at 28 °C. The expressed protein was accumulated in the soluble fraction of cell lysate. The molecular mass was determined on the basis of marker proteins on SDS-PAGE; it was about 110 kDa. GST was cleaved from the protein construct by incubation with thrombin for 12 h and the hCD98 was separated by Sephadex G-200 chromatography (size exclusion). hCD98 showed a 62 kDa apparent molecular mass, as determined on the basis of molecular mass markers using SDS-PAGE. The yield of CD98 was 2 mg/L of cell culture.     

The proteins cleaved by endogenous tryptic proteases in normal EDTA plasma by C18 collection of peptides for liquid chromatography micro electrospray ionization and tandem mass spectrometry

The tryptic peptides from ice cold versus room temperature plasma were identified by C18 liquid chromatography and micro electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS). Samples collected on ice showed low levels of endogenous tryptic peptides compared to the same samples incubated at room temperature. Plasma on ice contained peptides from albumin, complement, and apolipoproteins and others that were observed by the X!TANDEM and SEQUEST algorithms. In contrast to ice cold samples, after incubation at room temperature, greater numbers of tryptic peptides from well characterized plasma proteins, and from cellular proteins were observed. A total of 583,927 precursor ions and MS/MS spectra were correlated to 94,669 best fit peptides that reduced to 22,287 correlations to the best accession within a gene symbol and to 7174 correlations to at least 510 gene symbols with ≥ 5 independent MS/MS correlations (peptide counts) that showed FDR q-values ranging from E?9 (i.e. FDR = 0.000000001) to E?227. A set of 528 gene symbols identified by X!TANDEM and SEQUEST including C4B showed ≥ fivefold variation between ice cold versus room temperature incubation. STRING analysis of the protein gene symbols observed from endogenous peptides in normal plasma revealed an extensive protein-interaction network of cellular factors associated with cell signalling and regulation, the formation of membrane bound organelles, cellular exosomes and exocytosis network proteins. Taken together the results indicated that a pool of cellular proteins, or protein complexes, in plasma are apparently not stable and degrade soon after incubation at room temperature.     

Conserved structure and varied expression reveal key roles of phosphoglucan phosphatase gene starch excess 4 in barley

As one of the phosphoglucan phosphatases, starch excess 4 (SEX4) encoded by SEX4 gene has recently been intensively studied because of its vital role in the degradation of leaf starch. In this study, we isolated and chromosomally mapped barley SEX4, characterized its gene and protein structure, predicted the cis-elements of its promoter, and analysed its expression based on real-time quantitative PCR and publically available microarray data. The full length of barely SEX4 (HvSEX4) was 4,598 bp and it was mapped on the long arm of chromosome 4H (4HL). This gene contained 14 exons and 13 introns in all but two of the species analysed, Arabidopsis (13 exons and 12 introns) and Oryza brachyantha (12 exons and 11 introns). An exon–intron junction composed of intron 4 to intron 7 and exon 5 to exon 8 was highly conserved among the analysed species. SEX4 is characterized with conserved functional domains (dual specificity phosphatase domain and carbohydrate-binding module 48) and varied chloroplast transit peptide and C-terminal. Expression analyses indicated that: (1) SEX4 was mainly expressed in anthers of barley, young leaf and anthers of rice, and leaf of Arabidopsis; (2) it exhibited a diurnal pattern in barley, rice and Arabidopsis; (3) significant difference in the expression of SEX4 was not detected for either barley or rice under any of the investigated stresses; and (4) it was significantly down-regulated at middle stage and up-regulated at late stage under cold treatment, down-regulated at early stage under heat treatment, and up-regulated at late stage under salt treatment in Arabidopsis. The strong relationships detected in the current study between SEX4 and glucan, water dikinases (GWD) or phosphoglucan, water dikinases (PWD) were discussed. Collectively, our results provide insights into genetic manipulation of SEX4, especially in monocotyledon and uncovering the possible roles of SEX4 in plant development.     

An improved method and cost effective strategy for soluble expression and purification of human N-myristoyltransferase 1 in E. coli

N-myristoyltransferase (NMT) is an indispensible enzyme, which exists as two isoforms (NMT1 and NMT2) in humans and has proven roles in development of cancerous states. It is thus a target for novel anti-cancer drug design, but understanding of the biochemical and functional differences of these isozymes is not fully deciphered. A soluble expression under the T7 promoter for human NMT1 was achieved in E. coli BL21 (DE3) cells, devoid of any isopropyl β-d-1-thiogalactopyranoside-based induction. The identity of expressed protein was confirmed by matrix-assisted laser desorption ionization mass spectrometry peptide-fingerprint analysis and a two-step purification protocol yielded homogeneous enzyme. The intact mass of the purified protein was verified by electrospray ionization mass spectrometry and found to be in agreement with the theoretical mass (48.141 vs. 48.140 kDa). The fluorescence spectrophotometric analyses of the ligand binding and enzyme activity demonstrated that the recombinant form is functional. The yield of purified protein was ~8–10 mg/L culture (batch to batch variation) with a specific activity value of 18,500 ± 513 U/mg of protein under the experimental conditions used. The final verification of the myristoylation was demonstrated by mass spectrometry analysis of reaction product. The described approach could be readily adapted for production of human NMT1, with high yields of pure enzyme preparations, which should aid in downstream applications involving inhibitor design and structure–function studies of NMT’s.     

Detection of a phosphorylated glycine-serine linker in an IgG-based fusion protein

Molecular mass determination by electrospray ionization mass spectrometry of a recombinant IgG-based fusion protein (mAb1-F) produced in human embryonic kidney (HEK) cells demonstrated the presence of a dominant +79 Da product variant. Using LC-MS tryptic peptide mapping analysis and collision-induced dissociation (CID) and electron-transfer/higher-energy collision dissociation fragmentations, the modification was localized to the C-terminal serine residue of a glycine-serine linker [(G₄S)₂] of a fused heavy chain containing in total 2 (G₄S)₂-linkers. The modification was identified as a phosphorylation (+79.97 Da) by the presence of a 98 Da neutral loss reaction with CID, by spiking a synthetic phosphoserine peptide, and by dephosphorylation with alkaline phosphatase. A thermolysin digest combined with higher-energy collision dissociation (HCD) positioned the phosphoserine to one specific glycine-serine linker of the fused heavy chain, and the relative level of phosphorylated linker was determined to be 11.3% and 0.4% by LC-MS when the fusion protein was transiently expressed in HEK or in stably transformed Chinese hamster ovary cells, respectively. This observation demonstrates that fusions with glycine-serine linker sequences should be carefully evaluated during drug development to prevent the introduction of a phosphorylation site in therapeutic fusion proteins.     

New wheat-rye 5DS-4RS·4RL and 4RS-5DS·5DL translocation lines with powdery mildew resistance

Powdery mildew is one of the serious diseases of wheat (Triticum aestivum L., 2n = 6 × = 42, genomes AABBDD). Rye (Secale cereale L., 2n = 2 × = 14, genome RR) offers a rich reservoir of powdery mildew resistant genes for wheat breeding program. However, extensive use of these resistant genes may render them susceptible to new pathogen races because of co-evolution of host and pathogen. Therefore, the continuous exploration of new powdery mildew resistant genes is important to wheat breeding program. In the present study, we identified several wheat-rye addition lines from the progeny of T. aestivum L. Mianyang11 × S. cereale L. Kustro, i.e., monosomic addition lines of the rye chromosomes 4R and 6R; a disomic addition line of 6R; and monotelosomic or ditelosomic addition lines of the long arms of rye chromosomes 4R (4RL) and 6R (6RL). All these lines displayed immunity to powdery mildew. Thus, we concluded that both the 4RL and 6RL arms of Kustro contain powdery mildew resistant genes. It is the first time to discover that 4RL arm carries powdery mildew resistant gene. Additionally, wheat lines containing new wheat-rye translocation chromosomes were also obtained: these lines retained a short arm of wheat chromosome 5D (5DS) on which rye chromosome 4R was fused through the short arm 4RS (designated 5DS-4RS·4RL; 4RL stands for the long arm of rye chromosome 4R); or they had an extra short arm of rye chromosome 4R (4RS) that was attached to the short arm of wheat chromosome 5D (5DS) (designated 4RS-5DS·5DL; 5DL stands for the long arm of wheat chromosome 5D). These two translocation chromosomes could be transmitted to next generation stably, and the wheat lines containing 5DS-4RS·4RL chromosome also displayed immunity to powdery mildew. The materials obtained in this study can be used for wheat powdery mildew resistant breeding program.     

Heteromultimeric structure of the nitrate reductase complex of Chlamydomonas reinhardii

The NAD(P)H-nitrate reductase complex (overall-NR) of Chlamydomonas reinhardii exhibits two partial activities: NAD(P)H-cytochrome c reductase (diaphorase) and reduced benzyl viologen-NR (terminal-NR). Mild tryptic digestion of the enzyme complex resulted in the loss of both overall and terminal-NR activities, whereas diaphorase activity remained unaltered. The diaphorase activity of mutant 104 and the terminal-NR activity of mutant 305 of C. reinhardii, which are the sole activities related to NR present in these mutants, responded to tryptic treatment to the same extent as the corresponding activities of the wild enzyme complex. Trypsin disassembled the 220-kd NR native complex by destroying the aggregation capability of the diaphorase subunits without affecting their activity nor molecular size (45 kd). A 67-kd thermostable protein, containing molybdenum co-factor, was also released from trypsin-treated NR. This protein lacked diaphorase and NR activities but was able to reconstitute the overall-NR complex by complementation with untreated diaphorase subunit of mutant 104. Our results support a tetrameric structure for the C. reinhardii NR complex, containing two kinds of subunits.     

Sulfolobus tokodaii ST2133 is characterized as a thioredoxin reductase-like ferredoxin:NADP+ oxidoreductase

The putative gene (st2133) for ferredoxin:NADP⁺ oxidoreductase (FNR) from Sulfolobus tokodaii, a thermoacidophilic crenarchaeon, was heterologously expressed. About 90 % of the purified product was a homodimer containing 0.46 mol FAD/mol subunit, and showing NADPH:DCPIP oxidoreductase activity, V _max being 1.38 and 21.8 U/mg (70 °C) in the absence and presence of 1 mM FMN. NADPH was a much better electron donor than NADH with various electron acceptors, such as oxygen, hydrogen peroxide, DCPIP, cytochrome c, and dithiobisnitrobenzoate. Most of the reactions were activated by 15- to 140-fold on addition of FMN, while FAD was 5–10 times less effective. Ferredoxin (Fd) from S. tokodaii served as an electron carrier in both Fd-dependent NADPH formation and NADPH-dependent Fd reduction. ST2133 belongs to the thioredoxin reductase-like protein family, which is slightly distantly related to FNR family proteins from bacteria, plants and man. This is the first report on FNR from a crenarchaeon, providing a clue to the recycling of Fd during archaeal metabolism.     

Supramolecular structure of the OXPHOS system in highly thermogenic tissue of Arum maculatum

The protein complexes of the mitochondrial respiratory chain associate in defined ways forming supramolecular structures called respiratory supercomplexes or respirasomes. In plants, additional oxidoreductases participate in respiratory electron transport, e.g. the so-called “alternative NAD(P)H dehydrogenases” or an extra terminal oxidase called “alternative oxidase” (AOX). These additional enzymes were previously reported not to form part of respiratory supercomplexes. However, formation of respiratory supercomplexes might indirectly affect “alternative respiration” because electrons can be channeled within the supercomplexes which reduces access of the alternative enzymes towards their electron donating substrates. Here we report an investigation on the supramolecular organization of the respiratory chain in thermogenic Arum maculatum appendix mitochondria, which are known to have a highly active AOX for heat production. Investigations based on mild membrane solubilization by digitonin and protein separation by blue native PAGE revealed a very special organization of the respiratory chain in A. maculatum, which strikingly differs to the one described for the model plant Arabidopsis thaliana: (i) complex I is not present in monomeric form but exclusively forms part of a I + III₂ supercomplex, (ii) the III₂ + IV and I + III₂ + IV supercomplexes are detectable but of low abundance, (iii) complex II has fewer subunits than in A. thaliana, and (iv) complex IV is mainly present as a monomer in a larger form termed “complex IVa”. Since thermogenic tissue of A. maculatum at the same time has high AOX and I + III₂ supercomplex abundance and activity, negative regulation of the alternative oxidase by supercomplex formation seems not to occur. Functional implications are discussed.     

A root-specific wall-associated kinase gene, HvWAK1, regulates root growth and is highly divergent in barley and other cereals

Wall-associated receptor-like kinases (WAKs) are important candidates for directly linking the extracellular matrix with intracellular compartments and are involved in developmental processes and stress response. WAK gene family has been identified in plants such as Arabidopsis and rice. Here, we present a detailed analysis of the WAK1 gene from barley cv. Golden Promise, mapped to chromosome 5H. Three BAC clones corresponding to the WAK fragment were sequenced and the full-length WAK1 gene was characterized. The gene has three exons and two short introns with a coding region of 2,178 bp encoding a protein of 725 amino acids. A regulatory region was analyzed in ?1,000 bp sequence upstream to start codon. Using conserved domains database and SMART, various conserved domains such as GUB WAK Bind, epidermal growth factor CA, and protein kinase C as well as other regions like signal peptides, active sites, and transmembrane domains were identified. The gene organization of HvWAK1 was compared with wheat (TaWAK1) and Arabidopsis (AtWAK1), suggesting that the WAK1 gene organization has remained highly conserved. Nonetheless, WAK1 was found to be highly divergent when compared with sequences available from barley cv. Haruna Nijo (50 %), rice (46 %), wheat (21 %), Arabidopsis (25 %), and maize (19 %). This divergence may have facilitated a better adaptation to surrounding environments due to its role in communication between the extracellular matrix, cell, and outer environment. Semiquantitative RT-PCR-based expression analysis indicates HvWAK1 expression is specific to roots. Significant differences in root growth between GP wild type and GP-Ds mutant seedlings were observed under control and salt stress conditions.     

Arabidopsis nudix hydrolase 7 plays a role in seed germination

Arabidopsis nudix hydrolase 7 (Atnudt7) mutants exhibit reduced seed germination phenotype following after-ripening. The role of AtNUDT7 in seeds and during early stages of imbibition was examined. Seeds of Atnudt7-1 and Col-0 following 3 days of imbibition were used to profile changes in NADH- and ADP-ribose pyrophosphohydrolase enzyme activities, expression of nudix family genes closely related to AtNudt7, and AtNUDT7 protein levels. Changes in pyridine nucleotides, phytohormones, reactive oxygen species and poly(ADP-ribose) levels in after-ripened seeds and 1 day after imbibition were also analyzed. Changes in AtNUDT7 gene expression, protein levels and enzyme activities in WT seeds and during early stages of imbibition were correlated. Atnudt7-1 seeds lacked NADH pyrophosphohydrolase activity that led to very high catabolic redox charge. Abscisic acid (ABA) levels were higher in Atnudt7-1 mutant while salicylic acid, gibberellic acid, and reactive oxygen species (ROS) levels were higher in WT seeds. In Atnudt7-1, there was excess ROS accumulation 1 day after imbibition. PAR levels were significantly higher in Atnudt7-1 mutant when compared to WT during imbibition. Based on these observations, we conclude NADH pyrophosphohydrolase activity conferred by AtNUDT7 is important for NAD:NADH homeostasis in seeds. Perturbations to this key redox couple alter ABA and ROS levels in the seeds that in turn lowers germination.     

Ectopic overexpression of the aluminum-induced protein gene from Panax ginseng enhances heavy metal tolerance in transgenic Arabidopsis

Aluminum (Al), one of the most abundant metals in agricultural soils, significantly limits crop growth and productivity in acidic soil by inhibiting root elongation. Al ions, especially Al³⁺, have a toxic effect on both plant and animal cells under low-pH conditions. We first isolated and characterized aluminum-induced protein (AIP) cDNA from a 4-year-old root of Panax ginseng Meyer. This cDNA encodes an open reading frame of 711 bp with a deduced amino acid sequence of 236 residues. The calculated molecular mass of the mature protein is approximately 58.9 kDa with a predicated isoelectric point of 5.13. The Panax ginseng AIP (PgAIP) contains a domain also present in wheat aluminum-induced protein 7 (Wali7) and shares homology with the AIPs of other species, including Codonopis and Arabidopsis. The PgAIP gene was abundantly expressed in the plant’s leaves and was up-regulated by Al exposure. The functional role of PgAIP in Al tolerance was further validated through its overexpression in Arabidopsis. Transgenic Arabidopsis plants overexpressing the PgAIP gene showed enhanced Al tolerance in terms of root growth when compared to wild-type plants, suggesting PgAIP is important in plant defense against Al toxicity. Confocal analysis of CFP-tagging PgAIP in Arabidopsis showed subcellular localization in the plasma membrane. Our results suggest that PgAIP in the plasma membrane plays an important role in the protection of plant cells against heavy metal exposure.