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Publikation

Rekik, I.; Drira, N.; Grubb, C. D.; Elleuch, A.; Molecular characterization and evolution studies of a SERK like gene transcriptionally induced during somatic embryogenesis in Phoenix Dactylifera L v Deglet Nour Genetika 47, 323-337, (2015) DOI: 10.2298/GENSR1501323R

A somatic embryogenesis receptor kinase like (SERKL) cDNA, designated PhSERKL, was isolated from date palm (Phoenix Dactylifera L) using RACE PCR. PhSERKL protein shared all the characteristic domains of the SERK family, including five leucine-rich repeats, one proline-rich region motif, a transmembrane domain, and kinase domains. Phylogenetic analyses using PHYLIP and Notung 2.7 programs suggest that the SERK proteins of some plant species resulted from relatively ancient duplication events. We predict an ancestor protein of monocots and dicots SERK using FASTML program. Somatic embryogenic cultures of date palm were established following transfer of callus cultures to medium containing 2, 4-dichlorophenoxyacetic acid. The role of PhSERKL gene during establishment of somatic embryogenesis in culture was investigated using quantitative real-time PCR. PhSERKL gene was highly expressed during embryogenic competence acquisition and globular embryo formation in culture. Overall, levels of expression of PhSERKL gene were lower in nonembryogenic tissues and organs than in embryogenic callus.
Publikation

Rekik, I.; Chaâbene, Z.; Grubb, C. D.; Drira, N.; Cheour, F.; Elleuch, A.; In silico characterization and Molecular modeling of double-strand break repair protein MRE11 from Phoenix dactylifera v deglet nour Theor. Biol. Med. Model. 12, 23, (2015) DOI: 10.1186/s12976-015-0013-2

BackgroundDNA double-strand breaks (DSBs) are highly cytotoxic and mutagenic. MRE11 plays an essential role in repairing DNA by cleaving broken ends through its 3′ to 5′ exonuclease and single-stranded DNA endonuclease activities.MethodsThe present study aimed to in silico characterization and molecular modeling of MRE11 from Phoenix dactylifera L cv deglet nour (DnMRE11) by various bioinformatic approaches. To identify DnMRE11 cDNA, assembled contigs from our cDNA libraries were analysed using the Blast2GO2.8 program.ResultsThe DnMRE11 protein length was 726 amino acids. The results of HUMMER show that DnMRE11 is formed by three domains: the N-terminal core domain containing the nuclease and capping domains, the C-terminal half containing the DNA binding and coiled coil region. The structure of DnMRE11 is predicted using the Swiss-Model server, which contains the nuclease and capping domains. The obtained model was verified with the structure validation programs such as ProSA and QMEAN servers for reliability. Ligand binding studies using COACH indicated the interaction of DnMRE11 protein with two Mn2+ ions and dAMP. The ConSurf server predicted that residues of the active site and Nbs binding site have high conservation scores between plant species.ConclusionsA model structure of DnMRE11 was constructed and validated with various bioinformatics programs which suggested the predicted model to be satisfactory. Further validation studies were conducted by COACH analysis for active site ligand prediction, and revealed the presence of six ligands binding sites and two ligands (2 Mn2+ and dAMP).
Publikation

Hamdi, I.; Elleuch, A.; Bessaies, N.; Grubb, C. D.; Fakhfakh, H.; First report of Citrus viroid V in North Africa J. Gen. Plant Pathol. 81, 87-91, (2015) DOI: 10.1007/s10327-014-0556-9

We tested citrus samples from Tunisia using reverse transcription-polymerase chain reaction (RT-PCR), and for the first time, Citrus viroid V (CVd-V) was reported in North Africa. Fourteen of 38 tested citrus trees were infected by CVd-V including the majority of varieties grown in Tunisia. Some RT-PCR results were also supported by biological indexing. After sequencing the RT-PCR products, three new CVd-V variants were identified, showing 80–91 % nucleotide sequence identity with those reported previously. Based on phylogenetic analysis using all CVd-V sequences in GenBank, two main CVd-V groups were identified. Furthermore, construction of a genetic network of the detected haplotypes using the same sequences shows a clear geographical structuring of Tunisian CVd-V variants.
Publikation

Zayneb, C.; Bassem, K.; Zeineb, K.; Grubb, C. D.; Noureddine, D.; Hafedh, M.; Amine, E.; Physiological responses of fenugreek seedlings and plants treated with cadmium Environ. Sci. Pollut. Res. 22, 10679-10689, (2015) DOI: 10.1007/s11356-015-4270-8

The bioaccumulation efficiency of cadmium (Cd) by fenugreek (Trigonella foenum-graecum) was examined using different concentrations of CdCl2. The germination rate was similar to control except at 10 mM Cd. However, early seedling growth was quite sensitive to the metal from the lowest Cd level. Accordingly, amylase activity was reduced substantially on treatment of seeds with 0.5, 1, and 10 mM Cd. Cadmium also affected various other plant growth parameters. Its accumulation was markedly lower in shoots as compared to roots, reducing root biomass by almost 50 %. Plants treated with 1 and 5 mM Cd presented chlorosis due to a significant reduction in chlorophyll b especially. Furthermore, at Cd concentrations greater than 0.1 mM, plants showed several signs of oxidative stress; an enhancement in root hydrogen peroxide (H2O2) level and in shoot malondialdehyde (MDA) content was observed. Conversely, antioxidant enzyme activities (superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT)) increased in various plant parts. Likewise, total phenolic and flavonoid contents reached their highest values in the 0.5 mM Cd treatment, consistent with their roles in quenching low concentrations of reactive oxygen species (ROS). Consequently, maintaining oxidant and antioxidant balance may permit fenugreek to hyperaccumulate Cd and allow it to be employed in extremely Cd polluted soils for detoxification purposes.
Publikation

Zayneb, C.; Lamia, K.; Olfa, E.; Naïma, J.; Grubb, C. D.; Bassem, K.; Hafedh, M.; Amine, E.; Morphological, Physiological and Biochemical Impact of Ink Industry Effluent on Germination of Maize (Zea mays), Barley (Hordeum vulgare) and Sorghum (Sorghum bicolor) Bull. Environ. Contam. Toxicol. 95, 687-693, (2015) DOI: 10.1007/s00128-015-1600-y

The present study focuses on effects of untreated and treated ink industry wastewater on germination of maize, barley and sorghum. Wastewater had a high chemical oxygen demand (COD) and metal content compared to treated effluent. Germination decreased with increasing COD concentration. Speed of germination also followed the same trend, except for maize seeds exposed to untreated effluent (E), which germinated slightly faster than controls. These alterations of seedling development were mirrored by changes in soluble protein content. E exerted a positive effect on soluble protein content and maximum levels occurred after 10 days with treated effluent using coagulation/flocculation (TEc/f) process and treated effluent using combined process (coagulation/flocculation/biosorption) (TEc/f/b). Likewise, activity of α-amylase was influenced by effluent composition. Its expression depended on the species, exposure time and applied treatment. Nevertheless, current results indicated TEc/f/b had no observable toxic effects on germination and could be a beneficial alternative resource to irrigation water.
Publikation

Ziegler, J.; Qwegwer, J.; Schubert, M.; Erickson, J. L.; Schattat, M.; Bürstenbinder, K.; Grubb, C. D.; Abel, S.; Simultaneous analysis of apolar phytohormones and 1-aminocyclopropan-1-carboxylic acid by high performance liquid chromatography/electrospray negative ion tandem mass spectrometry via 9-fluorenylmethoxycarbonyl chloride derivatization J. Chromatogr. A 1362, 102-109, (2014) DOI: 10.1016/j.chroma.2014.08.029

A strategy to detect and quantify the polar ethylene precursor 1-aminocyclopropan-1-carboxylic acid (ACC) along with the more apolar phytohormones abscisic acid (ABA), indole-3-acetic acid (IAA), jasmonic acid (JA), jasmonic acid-isoleucine conjugate (JA-Ile), 12-oxo-phytodienoic acid (OPDA), trans-zeatin, and trans-zeatin 9-riboside using a single extraction is presented. Solid phase resins commonly employed for extraction of phytohormones do not allow the recovery of ACC. We circumvent this problem by attaching an apolar group to ACC via derivatization with the amino group specific reagent 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl). Derivatization in the methanolic crude extract does not modify other phytohormones. The derivatized ACC could be purified and detected together with the more apolar phytohormones using common solid phase extraction resins and reverse phase HPLC/electrospray negative ion tandem mass spectrometry. The limit of detection was in the low nanomolar range for all phytohormones, a sensitivity sufficient to accurately determine the phytohormone levels from less than 50 mg (fresh weight) of Arabidopsis thaliana and Nicotiana benthamiana tissues. Comparison with previously published phytohormone levels and the reported changes in phytohormone levels after stress treatments confirmed the accuracy of the method.
Publikation

Grubb, C. D.; Zipp, B. J.; Kopycki, J.; Schubert, M.; Quint, M.; Lim, E.-K.; Bowles, D. J.; Pedras, M. S. C.; Abel, S.; Comparative analysis of Arabidopsis UGT74 glucosyltransferases reveals a special role of UGT74C1 in glucosinolate biosynthesis Plant J. 79, 92-105, (2014) DOI: 10.1111/tpj.12541

The study of glucosinolates and their regulation has provided a powerful framework for the exploration of fundamental questions about the function, evolution, and ecological significance of plant natural products, but uncertainties about their metabolism remain. Previous work has identified one thiohydroximate S‐glucosyltransferase, UGT74B1, with an important role in the core pathway, but also made clear that this enzyme functions redundantly and cannot be the sole UDP‐glucose dependent glucosyltransferase (UGT) in glucosinolate synthesis. Here, we present the results of a nearly comprehensive in vitro activity screen of recombinant Arabidopsis Family 1 UGTs, which implicate other members of the UGT74 clade as candidate glucosinolate biosynthetic enzymes. Systematic genetic analysis of this clade indicates that UGT74C1 plays a special role in the synthesis of aliphatic glucosinolates, a conclusion strongly supported by phylogenetic and gene expression analyses. Finally, the ability of UGT74C1 to complement phenotypes and chemotypes of the ugt74b1‐2 knockout mutant and to express thiohydroximate UGT activity in planta provides conclusive evidence for UGT74C1 being an accessory enzyme in glucosinolate biosynthesis with a potential function during plant adaptation to environmental challenge.
Publikation

Kopycki, J.; Wieduwild, E.; Kohlschmidt, J.; Brandt, W.; Stepanova, A.; Alonso, J.; Pedras, M. S.; Abel, S.; Grubb, C. D.; Kinetic analysis of Arabidopsis glucosyltransferase UGT74B1 illustrates a general mechanism by which enzymes can escape product inhibition Biochem. J. 450, 37-46, (2013) DOI: 10.1042/BJ20121403

Plant genomes encode numerous small molecule glycosyltransferases which modulate the solubility, activity, immunogenicity and/or reactivity of hormones, xenobiotics and natural products. The products of these enzymes can accumulate to very high concentrations, yet somehow avoid inhibiting their own biosynthesis. Glucosyltransferase UGT74B1 (UDP-glycosyltransferase 74B1) catalyses the penultimate step in the core biosynthetic pathway of glucosinolates, a group of natural products with important functions in plant defence against pests and pathogens. We found that mutation of the highly conserved Ser284 to leucine [wei9-1 (weak ethylene insensitive)] caused only very mild morphological and metabolic phenotypes, in dramatic contrast with knockout mutants, indicating that steady state glucosinolate levels are actively regulated even in unchallenged plants. Analysis of the effects of the mutation via a structural modelling approach indicated that the affected serine interacts directly with UDP-glucose, but also predicted alterations in acceptor substrate affinity and the kcat value, sparking an interest in the kinetic behaviour of the wild-type enzyme. Initial velocity and inhibition studies revealed that UGT74B1 is not inhibited by its glycoside product. Together with the effects of the missense mutation, these findings are most consistent with a partial rapid equilibrium ordered mechanism. This model explains the lack of product inhibition observed both in vitro and in vivo, illustrating a general mechanism whereby enzymes can continue to function even at very high product/precursor ratios.
Publikation

Kopycki, J.; Schmidt, J.; Abel, S.; Grubb, C. D.; Chemoenzymatic synthesis of diverse thiohydroximates from glucosinolate-utilizing enzymes from Helix pomatia and Caldicellulosiruptor saccharolyticus Biotechnol. Lett. 33, 1039-1046, (2011) DOI: 10.1007/s10529-011-0530-y

Thiohydroximates comprise a diverse class of compounds important in both biological and industrial chemistry. Their syntheses are generally limited to simple alkyl and aryl compounds with few stereocenters and a narrow range of functional groups. We hypothesized that sequential action of two recombinant enzymes, a sulfatase from Helix pomatia and a β-O-glucosidase from Caldicellulosiruptor saccharolyticus, on glucosinolates would allow synthesis of thiohydroximates from a structurally broad array of abundant precursors. We report successful synthesis of thiohydroximates of varied chemical classes, including from homochiral compounds of demonstrated biological activity. The chemoenzymatic synthetic route reported here should allow access to many, if not all, of the thiohydroximate core structures of the ~200 known naturally occurring glucosinolates. The enrichment of this group for compounds with possible pharmacological potential is discussed.
Publikation

Grubb, C. D.; Abel, S.; Glucosinolate metabolism and its control Trends Plant Sci. 11, 89-100, (2006) DOI: 10.1016/j.tplants.2005.12.006

Glucosinolates and their associated degradation products have long been recognized for their distinctive benefits to human nutrition and plant defense. Because most of the structural genes of glucosinolate metabolism have been identified and functionally characterized in Arabidopsis thaliana, current research increasingly focuses on questions related to the regulation of glucosinolate synthesis, distribution and degradation as well as to the feasibility of engineering customized glucosinolate profiles. Here, we highlight recent progress in glucosinolate research, with particular emphasis on the biosynthetic pathway and its metabolic relationships to auxin homeostasis. We further discuss emerging insight into the signaling networks and regulatory proteins that control glucosinolate accumulation during plant development and in response to environmental challenge.
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