Claudia P. Spampinato

ANTI-SILENCING FUNCTION1 Proteins Are Involved in Ultraviolet-Induced DNA Damage Repair and Are Cell Cycle Regulated by E2F Transcription Factors in Arabidopsis

ASF1A and ASF1B genes are regulated by cell cycle progression and are involved in DNA repair after UV-B irradiation. ANTI-SILENCING FUNCTION1 (ASF1) is a key histone H3/H4 chaperone that participates in a variety of DNA- and chromatin-related processes, including DNA repair, where chromatin assembly and disassembly are of primary relevance. Information concerning the role of ASF1 proteins in the post-ultraviolet (UV) response in higher plants is currently limited. In Arabidopsis (Arabidopsis thaliana), an initial analysis of in vivo localization of ASF1A and ASF1B indicates that both proteins are mainly expressed in proliferative tissues. In silico promoter analysis identified ASF1A and ASF1B as potential targets of Elongation Factor2 (E2F) transcription factors. These observations were experimentally validated, both in vitro, by electrophoretic mobility shift assays, and in vivo, by chromatin immunoprecipitation assays and expression analysis using transgenic plants with altered levels of different E2F transcription factors. These data suggest that ASF1A and ASF1B are regulated during cell cycle progression through E2F transcription factors. In addition, we found that ASF1A and ASF1B are associated with the UV-B-induced DNA damage response in Arabidopsis. Transcript levels of ASF1A and ASF1B were increased following UV-B treatment. Consistent with a potential role in UV-B response, RNA interference-silenced plants of both genes showed increased sensitivity to UV-B compared with wild-type plants. Finally, by coimmunoprecipitation analysis, we found that ASF1 physically interacts with amino-terminal acetylated histones H3 and H4 and with acetyltransferases of the Histone Acetyl Transferase subfamily, which are known to be involved in cell cycle control and DNA repair, among other functions. Together, we provide evidence that ASF1A and ASF1B are regulated by cell cycle progression and are involved in DNA repair after UV-B irradiation.

Regulation of plant MSH2 and MSH6 genes in the UV-B-induced DNA damage response

Deleterious effects of UV-B radiation on DNA include the formation of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). These lesions must be repaired to maintain the integrity of DNA and provide genetic stability. Of the several repair systems involved in the recognition and removal of UV-B-induced lesions in DNA, the focus in the present study was on the mismatch repair system (MMR). The contribution of MutSα (MSH2-MSH6) to UV-induced DNA lesion repair and cell cycle regulation was investigated. MSH2 and MSH6 genes in Arabidopsis and maize are up-regulated by UV-B, indicating that MMR may have a role in UV-B-induced DNA damage responses. Analysis of promoter sequences identified MSH6 as a target of the E2F transcription factors. Using electrophoretic mobility shift assays, MSH6 was experimentally validated as an E2F target gene, suggesting an interaction between MMR genes and the cell cycle control. Mutations in MSH2 or MSH6 caused an increased accumulation of CPDs relative to wild-type plants. In addition, msh2 mutant plants showed a different expression pattern of cell cycle marker genes after the UV-B treatment when compared with wild-type plants. Taken together, these data provide evidence that plant MutSα is involved in a UV-B-induced DNA damage response pathway.

From bacteria to plants: a compendium of mismatch repair assays.

Mismatch repair (MMR) system maintains genome integrity by correcting mispaired or unpaired bases which have escaped the proofreading activity of DNA polymerases. The basic features of the pathway have been highly conserved throughout evolution, although the nature and number of the proteins involved in the mechanism vary from prokaryotes to eukaryotes and even between humans and plants. Cells deficient in MMR genes have been observed to display a mutator phenotype characterized by an increased rate in spontaneous mutation, instability of microsatellite sequences and illegitimate recombination between diverged DNA sequences. Studies of the mutator phenotype have demonstrated a critical role for the MMR system in mutation avoidance and genetic stability. Here, we briefly review our current knowledge of the MMR mechanism and then focus on the in vivo biochemical and genetic assays used to investigate the function of the MMR proteins in processing DNA mismatches generated during replication and mitotic recombination in Escherichia coli, Saccharomyces cerevisiae, Homo sapiens and Arabidopsis thaliana. An overview of the biochemical assays developed to study mismatch correction in vitro is also provided.

Metabolic profiles of soybean roots during early stages of Fusarium tucumaniae infection

Soybean germplasm exhibits various levels of resistance to Fusarium tucumaniae, the main causal agent of sudden death syndrome (SDS) of soybean in Argentina. In this study, two soybean genotypes, one susceptible (NA 4613) and one partially resistant (DM 4670) to SDS infection, were inoculated with F. tucumaniae. Disease symptoms were scored at 7, 10, 14, and 25 days post-inoculation (dpi). The greatest difference in the area under the disease progress curve (AUDPC) values among genotypes was observed at 25 dpi. In order to detect early metabolic markers that could potentially discriminate between susceptible and resistant genotypes, gas chromatography-mass spectrometry (GC-MS) analyses of root samples were performed. These analyses show higher levels of several amino acids and the polyamine cadaverine in the inoculated than in the uninoculated susceptible cultivar at 7 dpi. Principal component analysis (PCA) revealed that the metabolic profile of roots harvested at the earliest time points from the inoculated susceptible genotype was clearly differentiated from the rest of the samples. Furthermore, variables associated with the first principal component were mainly amino acids. Taken together, the results indicate that the pathogen induced the susceptible plant to accumulate amino acids in roots at early time points after infection, suggesting that GC-MS-based metabolomics could be used for the rapid characterization of cultivar response to SDS.

Studies of the mechanism of transgene integration into plant protoplasts : improvement of the transformation rate

SummaryThe production of transgenic plants by means of direct gene transfer to protoplasts is now a widely-used technique. The biological mechanisms underlying the transformation are still poorly understood, but many investigations have attempted to shed light on some components of this process. Varying the experimental conditions has in some cases led to better transformation rates, but further improvements of the protocols are possible. Such improvements will require a better understanding of how the alien DNA enters the cells, becomes integrated into the chromosomes and is treated as a part of the plant genome. Irradiation with sublethal doses of X-rays or UV-light has been shown to increase the transformation frequency, while certain drugs have been shown to act in a similar manner. The effects of these and other factors are discussed.

Analogues of NADP(+) as inhibitors and coenzymes for NADP(+) malic enzyme from maize leaves.

Structural analogues of the NADP+ were studied as potential coenzymes and inhibitors for NADP+ dependent malic enzyme from Zea mays L. leaves. Results showed that 1, N6-etheno-nicotinamide adenine dinucleotide phosphate (∈ NADP+), 3-acetylpyridine-adenine dinucleotide phosphate (APADP+), nicotinamide-hypoxanthine dinucleotide phosphate (NHDP+) and β-nicotinamide adenine dinucleotide 2′: 3′-cyclic monophosphate (2′3′NADPc+) act as alternate coenzymes for the enzyme and that there is little variation in the values of the Michaelis constants and only a threefold variation in Vmax for the five nucleotides. On the other hand, thionicotinamide-adenine dinucleotide phosphate (SNADP+), 3-aminopyridine-adenine dinucleotide phosphate (AADP+), adenosine 2′-monophosphate (2′AMP) and adenosine 2′: 3′-cyclic monophosphate (2′3′AMPc) were competitive inhibitors with respect to NADP+, while β-nicotinamide adenine dinucleotide 3′-phosphate (3′NADP+), NAD+, adenosine 3′-monophosphate (3′AMP), adenosine 2′: 5′-cyclic monophosphate (2′5′AMPc), 5′AMP, 5′ADP, 5′ATP and adenosine act as non-competitive inhibitors. These results, together with results of semiempirical self-consistent field-molecular orbitals calculations, suggest that the 2′-phosphate group is crucial for the nucleotide binding to the enzyme, whereas the charge density on the C4 atom of the pyridine ring is the major factor that governs the coenzyme activity.

Role of AtMSH7 in UV-B-induced DNA damage recognition and recombination

The mismatch repair (MMR) system maintains genome integrity by correcting replication-associated errors and inhibiting recombination between divergent DNA sequences. The basic features of the pathway have been highly conserved throughout evolution, although the nature and number of the proteins involved in this DNA repair system vary among organisms. Plants have an extra mismatch recognition protein, MutSγ, which is a heterodimer: MSH2-MSH7. To further understand the role of MSH7 in vivo, we present data from this protein in Arabidopsis thaliana. First, we generated transgenic plants that express β-glucuronidase (GUS) under the control of the MSH7 promoter. Histochemical staining of the transgenic plants indicated that MSH7 is preferentially expressed in proliferating tissues. Then, we identified msh7 T-DNA insertion mutants. Plants deficient in MSH7 show increased levels of UV-B-induced cyclobutane pyrimidine dimers relative to wild-type (WT) plants. Consistent with the patterns of MSH7 expression, we next analysed the role of the protein during somatic and meiotic recombination. The frequency of somatic recombination between homologous or homeologous repeats (divergence level of 1.6%) was monitored using a previously described GUS recombination reporter assay. Disruption of MSH7 has no effect on the rates of somatic homologous or homeologous recombination under control conditions or after UV-B exposure. However, the rate of meiotic recombination between two genetically linked seed-specific fluorescent markers was 97% higher in msh7 than in WT plants. Taken together, these results suggest that MSH7 is involved in UV-B-induced DNA damage recognition and in controlling meiotic recombination.

Mismatch recognition function of Arabidopsis thaliana MutSγ.

Genetic stability depends in part on an efficient DNA lesion recognition and correction by the DNA mismatch repair (MMR) system. In eukaryotes, MMR is initiated by the binding of heterodimeric MutS homologue (MSH) complexes, MSH2-MSH6 and MSH2-MSH3, which recognize and bind mismatches and unpaired nucleotides. Plants encode another mismatch recognition protein, named MSH7. MSH7 forms a heterodimer with MSH2 and the protein complex is designated MutSγ. We here report the effect the expression of Arabidopsis MSH2 and MSH7 alone or in combination exert on the genomic stability of Saccharomyces cerevisiae. AtMSH2 and AtMutSγ proteins failed to complement the hypermutator phenotype of an msh2 deficient strain. However, overexpressing AtMutSγ in MMR proficient strains generated a 4-fold increase in CAN1 forward mutation rate, when compared to wild-type strains. Can(r) mutation spectrum analysis of AtMutSγ overproducing strains revealed a substantial increase in the frequency of base substitution mutations, including an increased accumulation of base pair changes from G:C to A:T and T:A to C:G, G:C or A:T. Taken together, these results suggest that AtMutSγ affects yeast genomic stability by recognizing specific mismatches and preventing correction by yeast MutSα and MutSβ, with subsequent inability to interact with yeast downstream proteins needed to complete MMR.

Kinetic mechanism of NADP-malic enzyme from maize leaves.

The kinetic mechanism of NADP-dependent malic enzyme purified from maize leaves was studied in the physiological direction. Product inhibition and substrate analogues studies with 3′ aminopyridine dinucleotide phosphate and tartrate indicate that the enzyme reaction follows a sequential ordered Bi-Ter kinetic mechanism. NADP is the leading substrate followed by l-malate and the products are released in the order of CO2, pyruvate and NADPH. The enzyme also catalyzes a slow, magnesium-dependent decarboxylation of oxaloacetate and reduction of pyruvate and oxaloacetate in the presence of NADPH to produce l-lactate and l-malate, respectively.

Factors affecting the oligomeric state of NADP-malic enzyme from maize and wheat tissues: a chemical crosslinking study

The different aggregational states of maize and wheat NADP-malic enzyme as affected by pH, temperature and various metabolites have been studied by the combined use of intersubunit crosslinking and denaturing polyacrylamide gel electrophoresis. The association/dissociation equilibrium is a pH-dependent process: pH values above 8.0 promote the tetramer formation, while lowering the pH shifts the equilibria towards dimers and monomers. Below pH 6.0, most molecules exist as monomers. In the same way, the temperature governs the equilibria between the different oligomeric states. As the temperature is lowered from 42 to 0 degrees C, a progressive dissociation into dimers and monomers is observed. Excess enthalpies are negative in all cases, but the overall process demands an input of Gibbs free energy. Consequently, the protein dissociation is an entropy-driven process. The presence of Mg2+ or glycerol induces aggregation in both enzymes, while increasing the ionic strength produces the opposite effect. The results suggest that changes in the equilibria between monomer, dimer and tetramer of NADP-malic enzyme could be the molecular basis for an effective regulation of the enzyme activity in vivo.