Gene targeting in Arabidopsis thaliana.

Summary Gene targeting of a chromosomally integrated transgene in Arabidopsis thaliana is reported. A chimeric gene consisting of the promoter of the 35S RNA of CaMV, the polyadenylation signal of the octopine synthase gene and the coding region of the bacterial hygromycin phosphotransferase gene (hpt), which was rendered non-functional by deletion of 19 bp, was introduced into the genome of A. thaliana using Agrobacterium-mediated gene transfer. A total of 3.46 x 10⁸ protoplasts isolated from 17 independent transgenic Arabidopsis lines harbouring the defective chimeric hpt gene were transformed via direct gene transfer using various DNA forms containing only the intact coding region of the hpt gene. Out of 150 hygromycin-resistant colonies appearing in the course of these experiments, four were the result of targeted recombination of the incoming DNA with the defective chromosomal locus as revealed by PCR and Southern blot analysis. Comparison with the number of transformants obtained when an hpt gene controlled by a promoter and terminator from the nopaline synthase gene was employed results in a maximal ratio of homologous to non-homologous transformation in A. thaliana of 1 x 10^–4.     

Identification of Arabidopsis thaliana variants with differential glyphosate responses

To facilitate future investigations of glyphosate-resistance mechanisms, three approaches were taken to obtain Arabidopsis thaliana variants that differed in glyphosate response. Recurrent selection by spraying with sub-lethal glyphosate concentrations was performed with Columbia-0 seedlings. After seven cycles of treatment, no resistance was found. A population of 800,000 ethylmethanesulfonate-mutagenized M(2) seedlings was screened on agar containing 0.2mM glyphosate, a lower concentration than that previously used in other studies, and no resistant mutants were recovered. Seventy-two Arabidopsis ecotypes were screened with glyphosate and a range of responses was observed. In a follow-up experiment on a subset of these ecotypes, reduction of seed yield by 11.5 g/ha glyphosate (about 1% the typical field use rate) ranged among ecotypes from 0% to >90%, relative to untreated controls. However, even the least sensitive ecotypes were severely injured by relatively low glyphosate rates. Overall, attempts to select Arabidopsis seedlings that were significantly glyphosate-resistant were unsuccessful and consistent with previous reports. Arabidopsis ecotypes identified with differential glyphosate responses could be used for further studies though the inherently high sensitivity of Arabidopsis to glyphosate could limit their utility in studying glyphosate-resistance mechanisms.     

Proteins are polyisoprenylated in Arabidopsis thaliana

Isoprenoid lipids were found to be covalently linked to proteins of Arabidopsis thaliana. Their identity (polyprenols: Prenol-9-11 with Pren-10 dominating and dolichols: Dol-15-17 with Dol-16 dominating) was confirmed by means of HPLC/ESI-MS with application of the multiple reaction monitoring technique as well as metabolic labeling of Arabidopsis plants with [³H]mevalonate and other precursors. The occurrence of typical farnesol-, geranylgeraniol-, and phytol-modified proteins was also noted. Radioisotopic labeling allowed detection of several proteins that were covalently bound to mevalonate-derived isoprenoid alcohols. A significant portion of polyisoprenylated proteins was recovered in the cytosolic/light vesicular fraction of Arabidopsis cells upon subfractionation. Taken together our data prove that a subset of plant proteins is polyisoprenylated.     

Characterization of two Arabidopsis thaliana glutathione S-transferases

Glutathione S-transferases (GST) are multifunctional proteins encoded by a large gene family, divided on the basis of sequence identity into phi, tau, theta, zeta and lambda classes. The phi and tau classes are present only in plants. GSTs appear to be ubiquitous in plants and are involved in herbicide detoxification and stress response, but little is known about the precise role of GSTs in normal plant physiology and during biotic and abiotic stress response. Two cDNAs representing the two plant classes tau and phi, AtGSTF9 and AtGSTU26, were expressed in vitro and the corresponding proteins were analysed. Both GSTs were able to catalyse a glutathione conjugation to 1-chloro-2,4-dinitrobenzene (CDNB), but they were inactive as transferases towards p-nitrobenzylchloride (pNBC). AtGSTF9 showed activity towards benzyl isothiocyanate (BITC) and an activity as glutathione peroxidase with cumene hydroperoxide (CumHPO). AtGSTU26 was not active as glutathione peroxidase and towards BITC. RT-PCR analysis was used to evaluate the expression of the two genes in response to treatment with herbicides and safeners, chemicals, low and high temperature. Our results reveal that AtGSTU26 is induced by the chloroacetanilide herbicides alachlor and metolachlor and the safener benoxacor, and after exposure to low temperatures. In contrast, AtGSTF9 seems not to be influenced by the treatments employed.     

Homologues of a vacuolar processing enzyme that are expressed in different organs in Arabidopsis thaliana

Vacuolar processing enzymes (VPEs) are responsible for the maturation of seed proteins. These processing enzymes belong to a novel group of cysteine proteinases with molecular masses of 37 to 39 kDa. We isolated two genes of VPEs from a genomic library of Arabidopsis. The gene products were designated -VPE and -VPE, and they were 56% identical in terms of amino acid sequence. The amino acid sequences of -VPE and -VPE were also 55% and 67% identical to that of castor bean VPE, respectively. The gene for -VPE had 7 introns, while that of -VPE had 8 introns. Northern blot analysis revealed that -VPE is expressed in rosette leaves, cauline leaves and stems of Arabidopsis, while -VPE is predominantly expressed in the flowers and buds. Neither -VPE nor -VPE is expressed in the siliques. This result strongly suggests that the isolated genes encode isozymes of VPE that are specific to vegetative organs.     

Transient expression in leaf mesophyll protoplasts of Arabidopsis thaliana

Conditions for maximising transient expression of GUS in leaf mesophyll protoplasts of Arabidopsis thaliana ecotype C24 were investigated. It was found that the factors most influencing expression levels, with optimum levels in parenthesis, were plasmid DNA quantity (100 g per 5 × 10⁵ protoplasts), inclusion of carrier DNA (50 g), PEG pH and amount (pH above 6, and total PEG concentration at least 9% w/w) and the topological form of the DNA. Linearised plasmid DNA with long flanking sequences 3 and 5 to the marker gene yielded the highest levels of GUS expression.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - MU methylumbelliferone - PEG polyethylene glycol - X-gluc 5-bromo-4-chloro-3-indolyl--glucuronic acid     

Molecular characterization of multiple cDNA clones for ADP-glucose pyrophosphorylase from Arabidopsis thaliana

PCR amplification of cDNA prepared from poly(A)⁺ RNA from aerial parts of Arabidopsis thaliana, using degenerate nucleotide primers based on conserved regions between the large and small subunits of ADP-glucose pyrophosphorylase (AGP), yielded four different cDNAs of ca. 550 nucleotides each. Based on derived amino acid sequences, the identities between the clones varied from 49 to 69%. Sequence comparison to previously published cDNAs for AGP from various species and tissues has revealed that three of the amplified cDNAs (ApL1, ApL2 and ApL3) correspond to the large subunit of AGP, and one cDNA (ApS) encodes the small subunit of AGP. Both ApL1 and ApS were subsequently found to be present in a cDNA library made from Arabidopsis leaves. All four PCR products are encoded by single genes, as found by genomic Southern analysis.     

Expression and one-step purification of recombinant Arabidopsis thaliana frataxin homolog (AtFH)

Frataxin, a nuclear-encoded mitochondrial protein, has been proposed to participate in Fe-S cluster assembly, mitochondrial energy metabolism, respiration, and iron homeostasis. However, its precise function remains elusive. Frataxin is highly conserved in living organisms with no major structural changes, in particular at the C-terminal protein domain, suggesting that it plays a key function in all organisms. Recently, a plant gene, AtFH, with significant homology to other members of the frataxin family has been described. To gain insight on the frataxin role in plants, the frataxin domain was expressed in Escherichia coli BL21-codonPlus (DE3)-RIL cells and purified using a Ni-chelating column. The purified protein, added to a mixture containing Fe(II) and H2O2, attenuates the Fenton reaction indicating that the recombinant plant frataxin is functional. The procedure described here produced high yield of 99% pure protein through only one chromatographic step, suitable for further structure-function studies.     

A regulatory role for proline metabolism in stimulating Arabidopsis thaliana seed germination

Analysis of the rate-limiting dehydrogenases of the oxidative pentosephosphate pathway (OPPP), as well as changes in the contributions of¹⁴C₁- and ¹⁴C₆-labelled glucose torespired CO₂ during germination, indicated activation of the OPPPduring Arabidopsis seed germination. An approximatelyfour-fold increase in free proline (Pro) was also observed prior to radicleemergence in most seeds. Delayed radicle emergence in transgenicArabidopsis seeds which expressed an antisense copy of thegene encoding the Pro biosynthetic enzyme¹-pyrroline-5-carboxylate synthetase correlated with anapproximately 35% reduction in the maximal concentration of Pro accumulated bythe antisense lines during germination. A dose-dependent inhibition ofArabidopsis seed germination by millimolar concentrationsof Pro capable of feedback inhibition of Pro synthesis reinforced the role ofPro synthesis in promoting germination. The ability of the artificial oxidantsmethylene blue and phenazine ethosulphate to overcome the inhibitory effects ofPro suggests a functional link between elevated Pro synthesis and increasedOPPPactivity and the importance of coupling of both pathways in stimulatinggermination.     

Developmental anatomy of cotyledons and leaves in has mutant of Arabidopsis thaliana

Summary. In this work, we analyzed the developmental anatomy of cotyledons and leaves in the has mutant of Arabidopsis thaliana. It is a recessive T-DNA insertion mutation that causes changes in the size, shape, and tissue organization of the cotyledons and leaves of has plants. Analysis of has cotyledons revealed a prominent decrease in the cell number and an increase in the area of cotyledon cells and intercellular spaces of has plants. At early stages of development, has leaves are fingerlike structures, but later they develop small, lobed blades with rare trichomes. An important characteristic of the mutant leaf anatomy is the absence of mesophyll tissue differentiation. In addition, both cotyledons and leaves display a disrupted pattern of vascular bundles. Furthermore, mutant plants are defective in root and shoot morphology, indicating that the has mutation affects a number of aspects in plant development. Correspondence and reprints: Institute of Botany and “Jevremovac” Botanical Garden, Faculty of Biology, Belgrade University, Takovska 43, 11 000 Belgrade, Serbia.     

Hydrogen sulfide improves drought resistance in Arabidopsis thaliana

Hydrogen sulfide (H₂S) plays a crucial role in human and animal physiology. Its ubiquity and versatile properties have recently caught the attention of plant physiologists and biochemists. Two cysteine desulfhydrases (CDes), l-cysteine desulfhydrase and d-cysteine desulfhydrase, were identified as being mainly responsible for the degradation of cysteine in order to generate H₂S. This study investigated the expression regulation of these genes and their relationship to drought tolerance in Arabidopsis. First, the expression pattern of CDes in Arabidopsis was investigated. The expression levels of CDes gradually increased in an age-dependent manner. The expression of CDes was significantly higher in stems and cauline leaves than in roots, rosette leaves and flowers. Second, the protective effect of H₂S against drought was evaluated. The expression pattern of CDes was similar to the drought associated genes induced by dehydration, and H₂S fumigation was found to stimulate further the expression of drought associated genes. Drought also significantly induced increased H₂S production, a process that was reversed by re-watering. In addition, seedlings after treatment with NaHS (a H₂S donor) showed a higher survival rate and displayed a significant reduction in the size of the stomatal aperture compared to the control. These findings provide evidence that H₂S, as a gasotransmitter, improves drought resistance in Arabidopsis.     

Inhibition of CAT enzyme activity in Arabidopsis thaliana

Previously it was shown that transient chloramphenicol acetyltransferase (CAT) marker gene expression in Arabidopsis thaliana and Nicotiana tabacum resulted in significant differences in the accumulation of the CAT reaction products in radioactive CAT assays. Compared to Nicotiana tabacum, conversion of chloramphenicol to the acetylated products in Arabidopsis thaliana extracts was rather low. Here we report that the low CAT enzyme activity can be attributed in part to a heat sensitive CAT inhibitory effect in extracts of Arabidopsis thaliana. CAT enzyme activity in transgenic tobacco is inhibited by extracts from Arabidopsis. This inhibitory effect diminishes when Arabidopsis extracts were heat incubated. CAT activity in transgenic Arabidopsis lines was very low and was only detected in heat incubated extracts. Alternatively, enzyme-linked immunosorbent assays (ELISAs) can be used to detect the CAT protein in transgenic Arabidopsis.Abbreviations CAT chloramphenicol acetyltransferase - CAM chloramphenicol - ELISA enzyme linked immunosorbent assay     

Selection for enhanced germinal excision of Ac in transgenic Arabidopsis thaliana

Gene tagging in Arabidopsis thaliana using the autonomous Ac (Activator) transposable element has so far been hampered by low frequencies of germinal transposition events. Here we describe a procedure by which the frequency of independent germinal reinsertions has been much improved by a process of long-term selection on kanamycin for the continued growth of tissues in which somatic excisions have occurred. Growth on artificial media increased the somatic excision frequency, and the long-term selection procedure channelled somatic transposition events into the germline. This resulted in an overall germinal excision frequency in the progeny of longterm selected plants of 15%, as confirmed by Southern blotting, with 63% of the plants bearing excision events having detectable reinsertions of the Ac element. This compares with a germinal excision frequency of approximately 1% when no long-term selection is employed. However, offspring from individual plants tended to have identical germinal Ac reinsertion patterns, thus the critical parameter for evaluating the system for tagging purposes is the frequency of individual plants yielding offspring with reinsertions, which was 64%. This high frequency, when coupled to the enhanced germinal transposition rate overall, easily allows the generation of a large population of plants with independent reinsertions.