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.     

Isolation and characterization of an auxin-inducible glutathione S-transferase gene of Arabidopsis thaliana

Genes homologous to the auxin-inducible Nt103 glutathione S-transferase (GST) gene of tobacco, were isolated from a genomic library of Arabidopsis thaliana. We isolated a clone containing an auxin-inducible gene, At103-1a, and part of a constitutively expressed gene, At103-1b. The coding regions of the Arabidopsis genes were highly homologous to each other and to the coding region of the tobacco gene but distinct from the GST genes that have been isolated from arabidopsis thusfar. Overexpression of a cDNA clone in Escherichia coli revealed that the AT103-1A protein had GST activity.     

Proteins encoded by an auxin-regulated gene family of tobacco share limited but significant homology with glutathione S-transferases and one member indeed shows in vitro GST activity

A number of cDNAs corresponding to auxin-regulated mRNAs have been isolated from tobacco and found to be encoded by a multigene family consisting of three subfamilies. Homologous proteins have been isolated independently from soybean and potato. Here we report that the encoded proteins show a limited but significant homology to both plant and animal glutathione S-transferases (GST, EC 2.5.1.18). For the protein NT103, encoded by a member of the Nt103 subfamily, we demonstrate an in vitro GST activity. This is the first time a function is attributed to a member of this group of auxin-induced proteins or any of its homologues. The implications of this finding and the possible relationships between auxins and GSTs are discussed.     

A non-photosynthetic ferredoxin gene is induced by ethylene in Citrus organs

The sequence and expression of mRNA homologous to a cDNA encoding a non-photosynthetic ferredoxin (Fd1) from Citrus fruit was investigated. The non-photosynthetic nature of this ferredoxin was deduced from: (1) amino acid sequence alignments showing better scores with non-photosynthetic than with photosynthetic ferredoxins, (2) higher expression in tissues containing plastids other than chloroplast such as petals, young fruits, roots and peel of fully coloured fruits, and (3) the absence of light-dark regulation characteristic of photosynthetic ferredoxins. In a phylogenetic tree constructed with higher-plant ferredoxins, Citrus fruit ferredoxin clustered together with root ferredoxins and separated from the photosynthetic ferredoxins. Non photosynthetic (root and fruit) ferredoxins, but not the photosynthetic ferredoxins, have their closest homologs in cyanobacteria. Analysis of ferredoxin genomic organization suggested that non-photosynthetic ferredoxins exist in Citrus as a small gene family. Expression of Fd1 is developmentally regulated during flower opening and fruit maturation, both processes may be mediated by ethylene in Citrus. Exogenous ethylene application also induced the expression of Fd1 both in flavedo and leaves. The induction of non-photosynthetic ferredoxins could be related with the demand for reducing power in non-green, but biosynthetically active, tissues.     

A senescence-associated gene of Arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence

We have characterized the structure and expression of a senescence-associated gene (sen1) of Arabidopsis thaliana. The protein-coding region of the gene consists of 5 exons encoding 182 amino acids. The encoded peptide shows noticeable similarity to the bacterial sulfide dehydrogenase and 81% identity to the peptide encoded by the radish din1 gene. The 5-upstream region contains sequence motifs resembling the heat-shock- and ABA-responsive elements and the TCA motif conserved among stress-inducible genes. Examination of the expression patterns of the sen1 gene under various senescing conditions along with measurements of photochemical efficiency and of chlorophyll content revealed that the sen1 gene expression is associated with Arabidopsis leaf senescence. During the normal growth phase, the gene is strongly induced in leaves at 25 days after germination when inflorescence stems are 2–3 cm high, and then the mRNA level is maintained at a comparable level in naturally senescing leaves. In addition, dark-induced senescence of detached leaves or of leaves in planta resulted in a high-level induction of the gene. Expression of the sen1 gene was also strongly induced in leaves subjected to senescence by 0.1 mM abscisic acid or 1 mM ethephon treatment. The induced expression of the gene by dark treatment was not significantly repressed by treatment with 0.1 mM cytokinin or 50 mM CaCl₂ which delayed loss of chlorophyll but not that of photochemical efficiency.     

Altered development of Arabidopsis thaliana carrying the Agrobacterium tumefaciens ipt gene is partially due to ethylene effects

Transgenic Arabidopsis thaliana plants containingthe Agrobacterium tumefaciens cytokinin-biosynthesis geneipt were produced to study the effect of increasedcytokinin (CK) levels on the development of this rosette plant species. Inthreeindependently transformed lines (ipt-156, 158 and 161),Arabidopsis plants had smaller leaves, an underdevelopedroot system and decreased apical dominance in inflorescence stems. The smallertransgenic leaves were highly serrated along the margins, pale green and hadpointed leaf tips. In cross section, transgenic leaves had smaller cells andirregularly shaped epidermal cells. In the ipt-161 line,leaves and hypocotyls frequently exhibited purple color due to anthocyaninproduction. The most severe phenotype was observed in tissue cultureconditions,while growth in soil reduced or eliminated some phenotypic effects. Compared toC24 wild type plants, ipt-161 plants accumulated zeatinandzeatin riboside with an approximate 10-fold increase in the total pool of CKs.Astudy of the progeny resulting from crosses between theipt-161 transgenic line and the ethylene insensitivemutants ein1, ein2 andeti5 suggested that part of the altered developmentexhibited by the ipt transgenic plants was caused byincreased ethylene levels.     

Arabidopsis AtGSTF2 is regulated by ethylene and auxin, and encodes a glutathione S-transferase that interacts with flavonoids

Expression of the Arabidopsis glutathione S-transferase (GST) gene AtGSTF2 is induced by several stimuli, but the function of this GST remains unknown. We demonstrate that AtGSTF2 expression is also induced by glutathione, paraquat, copper, and naphthalene acetic acid (NAA) via a mechanism independent of ethylene perception, as determined by analysis of the ethylene-insensitive etr1 mutant. Deletion analyses identified two promoter regions important for regulation of AtGSTF2 expression in response to several of these inducers. Previous studies have suggested that AtGSTF2 interacts with indole-3-acetic acid (IAA) and the auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA). We show that recombinant AtGSTF2 directly binds IAA, NPA, and the artificial auxin NAA. As NPA may act as an endogenous flavonoid regulator of auxin transport, competition between NPA and flavonoids for binding to AtGSTF2 was examined. Both quercetin and kaempferol competed with NPA for AtGSTF2 binding, indicating that all three compounds bind AtGSTF2 at the same site. In transgenic Arabidopsis seedlings, AtGSTF2::GUS expression occurred at the root-shoot transition zone and was induced in this region, as well as at the root distal elongation zone, after treatment with IAA. In wild-type seedlings, AtGSTF2 is localized near the plasma membrane of cells in the root-shoot transition zone. However, both AtGSTF2::GUS expression and localization of AtGSTF2 protein were disrupted in flavonoid-deficient tt4 seedlings. Our results indicate that AtGSTF2 is involved not only in stress responses but also in development under normal growth conditions.     

Implication of two glutathione S-transferases in the optimal metabolism of m-toluate by Sphingomonas yanoikuyae B1

A putative glutathione S-transferase (GST) gene (bphK) was identified in the meta-cleavage operon for the degradation of m-toluate by Sphingomonas yanoikuyae B1. Disruption of bphK resulted in the loss of GST activity against 1-chloro-2,4-dinitrobenzene and a much increased lag time of the mutant strain MB3 (bphK::Km) following subculture into m-toluate medium. In contrast, an increased lag time was not observed when MB3 was grown on biphenyl or m-xylene and MB3 showed normal growth on m-toluate when complemented with a subclone containing the bphK gene only. Furthermore, an additional GST activity was detected in MB3. The induction timing of this second GST activity coincided with the beginning of the exponential growth phase of MB3 on m-toluate, reached maximal activity within three hours, and then dropped sharply to the basal level. Thus, it is apparent that BphK and/or the second GST are necessary for optimal growth of B1 on m-toluate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Two new oleosin isoforms with altered expression patterns in seeds of the Arabidopsis mutant fus3

Oleosins are proteins associated with lipid bodies mainly synthesised during seed development. Using a subtractive hybridisation approach two new members of the oleosin gene family of Arabidopsis thaliana have been isolated. The quantitative and temporal expression patterns of both genes are found to be affected in the fus3 mutant defective in late embryogenesis. This pattern is interpreted as a molecular marker for a mutant specific developmental change from a seed maturation toa germination pathway.     

Single-embryo RT-PCR assay to study gene expression dynamics during embryogenesis in<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

We have developed a single-embryo RT-PCR protocol for studying gene expression during plant embryogenesis. Four genes,glyceraldhyde-3-phosphate dehydrogenase (GAPC), shoot-meristemless (STM), monopteros (MP), andshaggy-like kinase etha (ASKη), fromArabidopsis thaliana were used to test the sensitivity and reliability of this method by analyzing the differential signal intensities of their RT-PCR products. The method could detect genes expressed during embryogenesis at a single-embryo level and, therefore, can be used to identify phenotypes. When in vitro, embryogenesis also is used to control the time course of zygote development exactly. The single-embryo RT-PCR protocol becomes a powerful method to survey the dynamics of specific gene expression.     

Phosphate deficiency-dependent upregulation of UDP-glucose pyrophosphorylase genes is insensitive to ABA and ethylene status in Arabidopsis leaves

The effects of inorganic phosphate (Pi) deficiency and ABA/ethylene status on expression of UDP-glucose pyrophosphorylase (UGPase) genes (Ugp), involved in sucrose/polysaccharide metabolism, were investigated. Both wild-type (wt), aba and abi mutants (ABA-deficient and -in-sensitive), etr, ein and eto (ethylene resistant and overproducing) grown on Pi-deficient and complete nutrient solution, as well as phol (Pi-deficient) mutants of Arabidopsis thaliana were used for experiments. Generally, Pi-deficiency conditions (including mannose feeding to decrease cytosolic Pi pool) resulted in an increase of Ugp expression in the leaves, under all experimental conditions. Mutant backgrounds reflecting differences in ABA or ethylene status/ sensitivity had no effect on the level of Ugp up-regulation by Pi-stress. Furthermore, feeding ABA to the leaves of wt and pho1 plants had no effect on Ugp expression, regardless of the sucrose status in the leaves. The data suggest that Pi deficiency leading to up-regulation of Ugp acts independently of ABA and ethylene status.     

The HKM gene, which is identical to the MS1 gene of Arabidopsis thaliana, is essential for primexine formation and exine pattern formation

A male-sterile mutant of Arabidopsis thaliana was isolated by T-DNA tagging screening. Using transmission electron microscopy analysis, we revealed that the microspores of this mutant did not have normal thick primexine on the microspore at the tetrad stage. Instead, a moderately electron-dense layer formed around the microspores. Although microspores without normal primexine failed to form a proper reticulate exine pattern at later stages, sporopollenin was deposited and an exine-like hackly structure was observed on the microspores during the microspore stage. Thus, this mutant was named hackly microspore (hkm). It is speculated that the moderately electron-dense layer was primexine, which partially played its role in sporopollenin deposition onto the microspore. Cytological analysis revealed that the tapetum of the hkm mutant was significantly vacuolated, and that vacuolated tapetal cells crushed the microspores, resulting in the absence of pollen grains within the anther at anthesis. Single nucleotide polymorphism analysis demonstrated that the hkm mutation exists within the MS1 gene, which has been reportedly expressed within the tapetum. Our results suggest that the critical process of primexine formation is under sporophytic control .     

Nucleotide sequence of a gene from Arabidopsis thaliana encoding a myb homologue

A gene encoding a proto-oncogene, a myb-related gene named Atmyb1, was cloned from Arabidopsis thaliana, and its nucleotide sequence was determined. The Atmyb1 gene contains an intron of 494 bp, and there are no highly homologous sequences present in the A. thaliana genome, but evidence was found that other myb-related genes exist. In the 5 flanking region, we found several typical cis-acting elements found in plant promoters. Sequence comparisons revealed that the ATMYB1 protein has a putative DNA-binding domain with two repeats of tryptophan clusters, which is common in MYB-related proteins in plants, while animal MYB-related proteins contain DNA-binding domains with three repeats of tryptophan clusters. The putative DNA-binding domain of the ATMYB1 protein has higher homology with that of the human c-MYB protein than with those of other plant MYB proteins.