Sublethal cadmium intoxication in Arabidopsis thaliana impacts translation at multiple levels.

To study the impact of translational regulation during heavy metal poisoning, Arabidopsis thaliana cell cultures were submitted to sublethal cadmium stress. At the concentration used, cadmium had a minimal impact on the growth of the culture but induced an accumulation of high molecular weight polysomes without de novo production of new ribosomes together with a reduction of protein synthesis. In addition, cadmium stress induces phosphorylation of eukaryotic initiation factor 2α by GCN2 and, in planta, gcn2 mutants are more sensitive to cadmium stress, suggesting a role for this translational regulation mechanism in the response to cadmium stress. Microarray analysis of total and polysomal RNAs in control and cadmium-treated cells reveals a large class of genes for which a variation in total RNA abundance is not linked to a variation in polysomal loading, suggesting that transcription and translation are uncoupled and that these genes are not recruited at the initiation step of translation.

Large-scale analysis of mRNA translation states during sucrose starvation in arabidopsis cells identifies cell proliferation and chromatin structure as targets of translational control.

Sucrose starvation of Arabidopsis (Arabidopsis thaliana) cell culture was used to identify translationally regulated genes by DNA microarray analysis. Cells were starved by subculture without sucrose, and total and polysomal RNA was extracted between 6 and 48 h. Probes were derived from both RNA populations and used to screen oligonucleotide microarrays. Out of 25,607 screened genes, 224 were found to be differentially accumulated in polysomal RNA following starvation and 21 were found to be invariant in polysomal RNA while their total RNA abundance was modified. Most of the mRNA appears to be translationally repressed (183/245 genes), which is consistent with a general decrease in metabolic activities during starvation. The parallel transcriptional analysis identifies 268 regulated genes. Comparison of transcriptional and translational gene lists highlights the importance of translational regulation (mostly repression) affecting genes involved in cell cycle and cell growth, these being overrepresented in translationally regulated genes, providing a molecular framework for the arrest of cell proliferation following starvation. Starvation-induced translational control also affects chromatin regulation genes, such as the HD1 histone deacetylase, and the level of histone H4 acetylation was found to increase during starvation. This suggests that regulation of the global nuclear transcriptional activity might be linked to cytoplasmic translational regulations.

Plant growth: the translational connection.

The TOR (target of rapamycin) pathway is a phylogenetically conserved transduction system in eukaryotes linking the energy status of the cell to the protein synthesis apparatus and to cell growth. The TOR protein is specifically inhibited by a rapamycin-FKBP12 complex (where FKBP stands for FK506-binding protein) in yeast and animal cells. Whereas plants appear insensitive to rapamycin, Arabidopsis thaliana harbours a single TOR gene, which is essential for embryonic development. It was found that the product of this gene was capable of binding to rapamycin and yeast FKBP12. In-frame fusion with a GUS reporter gene shows that the TOR protein is produced essentially in proliferating zones, whereas the TOR mRNA can be detected in all organs suggesting a translational regulation of TOR. Phenotypic analysis of Arabidopsis TOR mutants indicates that the plant TOR pathway fulfils the same role in controlling cell growth as its other eukaryotic counterparts.

Plant growth and the TOR pathway.

In mammalian, insect, and yeast cells, TOR proteins are essential regulators of cell growth in response to environmental signals including nutrients, mitogens, and stresses. Although many aspects of the TOR-dependent signalling pathway are conserved between animals and fungi, important differences have also been found and are likely to be related to the ecophysiological adaptations of these organisms. The TOR protein also exists in plants. This review will first discuss specific aspects of plants concerning the contribution of cell growth to overall growth, as well as their responses to nutrient starvation, with emphasis on recent results obtained through genetic analysis in the model plant Arabidopsis thaliana. This is followed by the current status of the genetic analysis of the TOR gene in this plant and the search for potential members of a TOR pathway in the Arabidopsis genome.

Utilization of mutants to analyze the interaction between Arabidopsis thaliana and its naturally root-associated Pseudomonas.

A model system based on the Arabidopsis thaliana (L.) Heynh. Ws ecotype and its naturally colonizing Pseudomonas thivervalensis rhizobacteria was defined. Pseudomonas strains colonizing A. thaliana were found to modify the root architecture either in vivo or in vitro. A gnotobiotic system using bacteria labelled with green fluorescent protein revealed that P. thivervalensis exhibited a colonization profile similar to that of other rhizobacterial species. Mutants of A. thaliana affected in root hair development and possible hormone perception were used to analyze the plant genetic determinants of bacterial colonization. A screen for mutants insensitive to P. thivervalensis colonization yielded two mutants found to be auxin resistant. This further supports a proposed role for bacterial auxin in inducing morphological modifications of roots. This work paves the way for studying the interaction between plants and non-pathogenic rhizobacteria in a gnotobiotic system, derived from a natural association, where interactions between both partners can be genetically dissected.

Plant lipoxygenase 2 is a translation initiation factor-4E-binding protein.

The eukaryotic initiation factor 4E (eIF4E) emerged recently as a target for different types of regulation affecting translation. In animal and yeast cells, eIF4E-binding proteins modulate the availability of eIF4E. A search for plant eIF4E-binding proteins from Arcabictopsis thaliana using the yeast genetic interaction system identified a clone encoding a lipoxygenase type 2 (AtLOX2). In vitro and in vivo biochemical assays confirm an interaction between AtLOX2 and plant eIF4E(iso) factor. A two-hybrid assay revealed that AtLOX2 is also able to interact with both wheat initiation factors 4E and 4E(iso). Deletion analysis maps the region of AtLOX2 involved in interaction with AteIF(iso)4E between amino acids 175 and 232. A sequence related to the conserved motif present in several eIF4E-binding proteins was found in this region. Furthermore, the wheat p86 subunit, a component of the plant translation eIF(iso)4F complex, was found to interfere with the AteIF(iso)4E-AtLOX2 interaction suggesting that p86 and AtLOX2 compete for the same site on eIF(iso)4E. These results may reflect a link between eIF4Es factors mediating translational control with LOX2 activity, which is probably conserved throughout the plant kingdom.

Identification of an Arabidopsis thaliana mutant accumulating threonine resulting from mutation in a new dihydrodipicolinate synthase gene.

A novel Arabidopsis DHDPS gene named DHDPS2 was found through identification of a mutant by promoter trapping. The mutation promotes a reduction of growth resulting from combination of a defect in lysine biosynthesis and accumulation of a toxic level of threonine or derived products. The mutant also modifies the amino acid composition issuing from the pyruvate and aspartate pathways, affecting mainly the root compartment. These data are in accordance with the expression of DHDPS2 in the root apex as visualized by expression of the GUS reporter gene. This suggests that a large proportion of the amino acids derived from pyruvate and aspartate are synthesized in this organ.

RFLP mapping of the whole genome of ten viral isolates representative of different biological groups of potato virus Y.

Ten PVY isolates representative of four PVY groups (YN, YNTN, YN-W, YO), differing by their ability to induce reactions of vein necrosis on tobacco and tuber necrosis on potato, were studied in order to research the regions of the viral genome involved in these necrosis phenomena. The whole genome of these isolates was amplified in two fragments (4,063 and 5,670 nucleotides) and was subjected to a restriction fragment length polymorphism (RFLP) study. In the first 4,063 nucleotides of the PVY genome, a phenetic analysis of RFLP data resulted in a clustering of our PVY isolates into three groups: PVYN isolates (group A); PVYNTN and PVYN-W isolates (group B) and PVYO isolates (group C). In the last 5,670 nucleotides, two groups were found: PVYN and PVYNTN isolates (group D) and PVYO and PVYN-W isolates (group E). From this clustering and the necrosing properties known for these isolates, the tobacco necrosis determinants seem more likely located in the 5' than in the 3' half part of the viral RNA, whereas it would be the opposite situation for the determinants of the necrosis on potato tubers. Moreover a recombination event seemed to have occurred in the genome of the PVYN-W isolates.

The Arabidopsis thaliana cDNAs coding for eIF4E and eIF(iso)4E are not functionally equivalent for yeast complementation and are differentially expressed during plant development.

Two cDNAs (At.EIF4E1 and At.EIF4E2) encoding, respectively, the eukaryotic initiation factors eIF4E and eIF(iso)4E of Arabidopsis thaliana were isolated by complementation of a Saccharomyces cerevisiae conditional mutant. The deduced amino acid sequences of the proteins are homologous to those from monocotyledonous plants, yeast and mammals. The corresponding genes were identified in YAC clones mapping to chromosome IV (At.EIF4E1) and to chromosome V (At.EIF4E2). The yeast strain complemented by At.EIF4E2 grew poorly compared with an isogenic strain expressing At.EIF4E1. Northern and in situ hybridization analysis show that both Arabidopsis At.EIF4E1 and At.EIF4E2 mRNAs are differentially accumulated in plant tissues. The At.EIF4E1 mRNA is expressed in all tissues except in the cells of the specialization zone of the roots; the At.EIF4E2 mRNA is particularly abundant in floral organs and in young developing tissues. This work further demonstrates an association between a high level of EIF4E mRNAs and cell proliferation and suggests that the plant eIF4E isoforms may have distinct functions in cell development and metabolism.

Synthesis of full-length potyvirus cDNA copies suitable for the analysis of genome polymorphism.

New methods facilitating the synthesis and amplification of full-length cDNA copies of single-stranded viral RNA genomes have been developed. A method is described for the efficient purification of potyviral RNA and total RNA from infected plants and it is shown that they can serve as templates for the efficient synthesis of a full-length, 10 kb long, genomic cDNA. Two different reverse transcriptases were used (AMV-RT and MMLV-RT); only the first reverse transcriptase produced a good quality, full-length cDNA using viral RNA as a template. Surprisingly, MMLV-RT allowed for the full-length cDNA synthesis on virions rather than viral RNA. The PVY cDNA, synthesized using either RNA or virions, can be amplified successfully by PCR with high yields of full-length products. Such products are good substrates for the study by RFLP of the total genome polymorphism of virus isolates.

Tobacco veinal necrosis determinants are unlikely to be located within the 5' and 3' terminal sequences of the potato virus Y genome.

Three potato virus Y isolates, representatives of distinct PVY groups, identified in potato fields in northern Poland were submitted to biological and molecular analysis. Phenotypically, two isolates, PVYN-Ny and PVYN-Wi, belong to the necrotic strain and the third one (PVYO-LW) to the common strain. PVYN-Wi, however, did not react with monoclonal antibodies directed against the necrotic strain isolates which recognise PVYN-Ny. To characterise the isolates, coat protein genes were sequenced and compared with sequences from databases. The necrotic PVYN-Wi isolate showed 99% amino acid homology with the common one-PVYO-LW and significantly differed from the second necrotic isolate (PVYN-Ny). Sequence based homology matrix and phylogenetic analysis lead to classification of PVYN-Ny into group I, encompassing solely necrotic strain isolates, whereas PVYN-Wi falls into a phenotypically heterogeneous group II. The sequence analysis allowed for identification of putative group I-specific epitopes. 3'NTR (non-translated region) sequences were identical for PVYN-Wi and PVYO-LW. The 5'NTR, P1 gene, coat protein gene and 3'NTR sequences of the common (PVYO-LW) and the necrotic (PVYN-Wi) isolates are 99-100% homologous. This suggests that tobacco veinal necrosis determinants are located outside the 3' and 5' terminal sequences of the PVY genome.

Switches in the mode of transmission select for or against a poorly aphid-transmissible strain of potato virus Y with reduced helper component and virus accumulation.

A poorly aphid-transmissible potato virus Y (PVY-PAT) variant emerged after several cycles of mechanical transmission of an initially aphid-transmissible (AT) isolate. Sequence analysis of the N-terminal region of the helper component-proteinase (HC-Pro) gene revealed a Lys to Glu change at a position previously found to abolish the HC-Pro aphid transmission activity in several potyviruses. Two cycles of aphid transmission allowed the virus population to evolve towards an AT form (PVY-ATnew) where a Glu to Lys change was observed. PVY-PAT produced lower amounts of coat protein and the accumulation of its HC-Pro in infected plants decreased from 7 to 28 days post-inoculation, as compared to PVY-ATnew. RT-PCR and restriction analysis showed that the two virus populations co-existed in the PVY-AT isolate and that the AT form was counter-selected during mechanical transmission. These observations suggest that the Lys to Glu substitution leads to decreased stability of HC-Pro resulting in poor transmissions by aphids, and further strengthen the idea that HC-Pro is involved in the accumulation of potyvirus in infected plants.

Cell-free cloning and biolistic inoculation of an infectious cDNA of potato virus Y.

Potato virus Y (PVY) full-length cDNA has been found to be refractory to cloning in Escherichia coli cells. A full-length 9.7 kb PVY cDNA was obtained by reverse transcription polymerase chain reaction (RT-PCR) from the RNA of PVY (tuber necrotic strain, PVYNTN). Double-stranded DNA fragments were used as primers (ds megaprimers), to include signals for transcription in vivo (a cauliflower mosaic virus 35S RNA promoter and a nopaline synthase terminator) in the final PCR product. Biolistic bombardment with a helium particle gun was used to inoculate the amplified product to detached tobacco leaves. Inoculation of tobacco plants with ground inoculated leaves followed by northern blot, ELISA and immuno-electron microscopy demonstrated that the DNA was highly infectious with up to 90% of bombarded leaves containing the virus. This methodology will allow the use of reverse genetics in the study of PVY-plant interactions and will also be useful for obtaining infectious cDNA from other viruses with large RNA genomes.

Sequence polymorphism in the 5'NTR and in the P1 coding region of potato virus Y genomic RNA.

Potato virus Y (PVY) the type member of the genus Potyvirus, occurs world-wide as isolates which differ in host range and the type of symptoms caused. The sequences of a 5' segment of viral RNA overlapping the 5' non-translated region (5'NTR) alone (ten isolates) or the 5'NTR and the adjacent P1 coding region (eight isolates) were established. These data were used to quantify the polymorphism in the 5'-terminal part of the PVY genome. Nucleotide sequence identity between isolates ranged from 66-100% in the 5'NTR and from 70-100% in the P1 coding region. The lowest amino acid sequence similarity between PVY P1 was 77%, illustrating the high variability of this protein in the PVY species. Phylogenetic trees based on either 5'NTR or P1 sequence analyses resulted in the same clustering of the studied isolates into three groups. Group I comprises potato isolates all inducing 'tobacco veinal necrosis' symptoms. Group II contains isolates inducing either 'tobacco veinal necrosis' or mosaic symptoms in tobacco. Group III contains mainly pepper or tomato isolates inducing mosaic symptoms in tobacco and shows a geographical clustering of the Tunisian isolates. This clustering into three groups is discussed in comparison with phylogenetic trees previously obtained from capsid gene or 3'NTR sequence analysis in the PVY species. Multiple sequence alignment indicated conserved motifs potentially involved in viral functions.

Sequence variability in the genome-3'-terminal region of BYDV for 10 geographically distinct PAV-like isolates of barley yellow dwarf virus: analysis of the ORF6 variation.

Nucleotide sequence of the genome terminal region 3' to the capsid-readthrough cistron were compared for 10 PAV-like isolates of barley yellow dwarf virus (BYDV) from three continents. The sequenced region varied in length from 853 to 864 nucleotides and the extent of sequence homology among the isolates ranged from 84 to 99%. Sequence variations occur mainly in two locations, one in the ORF6 coding region and the other near the genome 3' terminus. Sequence homology grouping reveals three genetically distinct clusters of PAV isolates (A, B and C). Cluster A consists of the Australian isolates, cluster B of one Canadian and three French isolates, and cluster C of the French isolate, RG. Dissimilarities with the corresponding genome-3'- terminal region of the BYDV-MAV serotype were greater than those observed between the PAV isolates alone. Comparison with the sequence of the 3' untranslated region of soybean dwarf virus revealed two stretches of nucleotide similarity, suggesting a common ancestor. Study of the coding ability revealed that the ORF6 is present in all the sequenced PAV isolates but differs in size and deduced amino acids composition. However, the fact that the majority of nucleotide changes are restricted to the third base position of the ORF6-codons suggests that ORF6 codes for a functional protein.

Mutations in zucchini yellow mosaic virus helper component protein associated with loss of aphid transmissibility.

Zucchini yellow mosaic virus (ZYMV) is a potyvirus transmitted by aphids in a non-persistent manner. Isolates having partially or totally lost their ability to be transmitted by aphids have been isolated and found to be affected in their helper component activities. We have sequenced the helper component coding region of poorly aphid-transmissible (PAT) variants of two strains of ZYMV, E15 and R5A. Mutations have been identified at the nucleotide level leading to two amino acid changes in the E15 PAT variant helper component and to one amino acid change located in the cysteine-rich region (well-conserved among potyviruses) in R5A PAT variant helper component. The mutation in the R5A variant changes the same amino acid as the one identified in potato virus C, a non-transmissible strain of potato virus Y.

Evolution and replication of tobacco ringspot virus satellite RNA mutants.

The replication properties of linker insertion-deletion mutants of tobacco ringspot virus satellite RNA have been studied by amplification in plants infected with the helper virus. Sequence analysis of the cDNAs corresponding to the replicated forms shows that only one of the original mutated molecules replicates unaltered, and in general new variants accumulate. Depending on the location of the original mutation three types of sequence modifications were observed: (i) deletion of the mutated region followed by sequence duplication, (ii) sequence duplication and deletion outside of the mutated region and (iii) limited rearrangements at the site of mutation. The mutant that replicates without sequence changes accumulates linear multimeric forms suggesting that self-cleavage is affected although the sequence alteration does not involve the hammerhead catalytic domain. Alternative RNA conformations are likely to play a role in the origin of this phenotype and in the formation of sequence duplications. These results demonstrate the great structural flexibility of this satellite RNA.

Nucleotide sequence of potato virus Y (N Strain) genomic RNA.

The complete nucleotide sequence of the genomic RNA of the potyvirus potato virus Y strain N (PVYn) was obtained from cloned cDNAs. This sequence is 9704 nucleotides long and can encode a polyprotein of 3063 amino acids. The positions of the cleavage sites at the N terminus of the capsid and cytoplasmic inclusion proteins have been determined. Other putative protein cleavage sites have been deduced by searching for consensus sequences and by analogy with the polyprotein of the tobacco vein mottling virus and of the tobacco etch virus. Comparison of the PVY polyprotein sequence with that of other potyvirus polyproteins shows similarities in genome organization and a high level of identity along most of the polyprotein, except for the putative proteins flanking the helper component. A search for specific protein motifs has revealed the existence of a potential metal-binding site at the putative N terminus of the helper component in potyviruses. The possible functions of this structure are discussed.

Expression vectors based on the Agrobacterium rhizogenes Ri plasmid transformation system.

This article describes several new expression vectors that capitalize on the ability of Agrobacterium rhizogenes to transfer DNA from its Ri plasmid to the plant nuclear genome. The intermediate vectors described include an expression cassette based on one of the three following promoters: the nopaline synthase promoter, or the cauliflower mosaic virus (CaMV) promoters responsible for transcription of either the 19S or 35S CaMV RNA. The termination and polyadenylation signals are either from the nopaline synthase gene or from CaMV. The expression micro-Ri plasmid described bears a selectable marker gene and an expression cassette cloned between the borders of the TL-region of the Ri plasmid of A. rhizogenes A4. Different strategies for using these vectors to introduce chimeric genes into plants are described, and the advantages and disadvantages of the two types of vectors are discussed.

lacZ gene fusions and insertion mutagenesis in the TL-region of Agrobacterium rhizogenes Ri plasmid.

Agrobacterium rhizogenes induces root formation and inserts a fragment of its plasmid into the genome of infected plants. A part of the transferred region (TL-region) of the Ri plasmid of A. rhizogenes strain A4 was cloned in pBR322. Insertions of the Escherichia coli lacZ coding region into the hybrid plasmids were made in vivo using mini-Mu-duction. Two mini-Mus were used, one with the Mu A and B transposase genes (MudII1681) and the other without (MudII1734). Two inserts which result in E. coli lacZ expression where shown to be located in the T-DNA region. This indicates that portions of the T-DNA are capable of expression in bacteria. When these two hybrid plasmids were transformed into Agrobacterium only the one harboring MudII1734 insert gave transformants which correspond to homologous recombination. These results indicate that gene fusion and insertion directed mutagenesis can be simultaneously obtained with this mini-Mu and could be used to study Agrobacterium gene expression.