Plant-specific environmental and developmental signals regulate the mismatch repair protein MSH6 in Arabidopsis thaliana.

The DNA mismatch repair (MMR) is a postreplicative system that guarantees genomic stability by correcting mispaired and unpaired nucleotides. In eukaryotic nuclei, MMR is initiated by the binding of heterodimeric MutS homologue (MSH) complexes to the DNA error or lesion. Among these proteins, MSH2-MSH6 is the most abundant heterodimer. Even though the MMR mechanism and proteins are highly conserved throughout evolution, physiological differences between species can lead to different regulatory features. Here, we investigated how light, sugar, and/or hormones modulate Arabidopsis thaliana MSH6 expression pattern. We first characterized the promoter region of MSH6. Phylogenetic shadowing revealed three highly conserved regions. These regions were analyzed by the generation of deletion constructs of the MSH6 full-length promoter fused to the ß-glucuronidase (GUS) gene. Combined, our in silico and genetic analyses revealed that a 121-bp promoter fragment was necessary for MSH6 expression and contained potential cis-acting elements involved in light- and hormone-responsive gene expression. Accordingly, light exposure or sugar treatment of four-day old A. thaliana seedlings triggered an upregulation of MSH6 in shoot and root apical meristems. Appropriately, MSH6 was also induced by the stem cell inducer WUSCHEL. Further, the stimulatory effect of light was dependent on the presence of phyA. In addition, treatment of seedlings with auxin or cytokinin also caused an upregulation of MSH6 under darkness. Consistent with auxin signals, MSH6 expression was suppressed in the GATA23 RNAi line compared with the wild type. Our results provide evidence that endogenous factors and environmental signals controlling plant growth and development regulate the MSH6 protein in A. thaliana.

Deficiency of the Arabidopsis mismatch repair MSH6 attenuates Pseudomonas syringae invasion.

The MutS homolog 6 (MSH6) is a nuclear DNA mismatch repair (MMR) gene that encodes the MSH6 protein. MSH6 interacts with MSH2 to form the MutSα heterodimer. MutSα corrects DNA mismatches and unpaired nucleotides arising during DNA replication, deamination of 5-methylcytosine, and recombination between non-identical DNA sequences. In addition to correcting DNA biosynthetic errors, MutSα also recognizes chemically damaged DNA bases. Here, we show that inactivation of MSH6 affects the basal susceptibility of Arabidopsis thaliana to Pseudomonas syringae pv tomato DC3000. The msh6 T-DNA insertional mutant exhibited a reduced susceptibility to the bacterial invasion. This heightened basal resistance of msh6 mutants appears to be dependent on an increased stomatal closure, an accumulation of H2O2 and double-strand breaks (DSBs) and a constitutive expression of pathogenesis-related (NPR1 and PR1) and DNA damage response (RAD51D and SOG1) genes. Complementation of this mutant with the MSH6 wild type allele under the control of its own promoter resulted in reversal of the basal bacterial resistance phenotype and the stomatal closure back to wild type levels. Taken together, these results demonstrate that inactivation of MSH6 increases Arabidopsis basal susceptibility to the bacterial pathogen and suggests a link between DNA repair and stress signaling in plants.

Role of the mismatch repair protein MSH7 in Arabidopsis adaptation to acute salt stress.

DNA mismatch repair (MMR) is a highly conserved pathway in evolution responsible for maintaining genomic stability. MMR is initiated when MutS proteins recognize and repair single base-base mismatches and small loops of unpaired nucleotides as well as certain types of DNA damage. Arabidopsis thaliana and other plants contain MutS protein homologs (MSH) found in other eukaryotic organisms and a unique MSH7 polypeptide. In this study, we first evaluated transient expression profiles of ten-days old pAtMSH7:GUS transgenic seedlings at different recovery times after an acute treatment for 48 hs with100 mM NaCl. GUS histochemical staining indicated that MSH7 expression is repressed by salt exposure but recovers progressively. Then, ten-days old mutants harboring two independent msh7 alleles were exposed for 48 hs with100 mM NaCl and different traits were measured over recovery time. Salt treated msh7 seedlings were defective in G2/M arrest. As a result, msh7 seedlings showed a reduced salt inhibitory effect as evidenced by a decreased reduction of rosette and leaf areas, stomatal density, total leaf number, silique length and seed number per silique. These findings suggest that disruption of MSH7 activity could be a promising approach for plant adaptive responses to salinity stress.

The mismatch repair protein MSH6 regulates somatic recombination in Arabidopsis thaliana.

The mismatch repair (MMR) pathway promotes genome stability by controlling the fidelity of replication and recombination. The first step of the pathway involves recognition of the mismatch by heterodimers composed of MutS homologs (MSH). Although MSH6 has been well characterized in yeasts and humans, the role of the plant protein has not been extensively studied. We first analyzed gene expression in Arabidopsis thaliana. The use of transgenic plants expressing the ß-glucuronidase (GUS) reporter gene under the control of approximately 1-kb region upstream of the start codon of the AtMSH6 gene demonstrated that MSH6 is preferentially expressed in undifferentiated cells with an intense cell division rate. We then examined protein function in meiotic and somatic recombination. Suppression of AtMSH6 did not affect the rate of meiotic recombination, but increased the frequency of recombination between two homeologous repeats of a marker gene by 3-fold relative to wild-type plants. Expression of the AtMSH6 gene under the control of its own promoter in msh6 homozygous mutant plants rescued the altered somatic recombination phenotype. We conclude that MSH6 shows a functional conservation across different biological kingdoms and a functional specificity in plants.

Growth and development of AtMSH7 mutants in Arabidopsis thaliana.

DNA mismatch repair (MMR) is a highly conserved biological pathway that improves the fidelity of DNA replication and recombination. MMR is initiated when MutS proteins recognize mismatches and small loops of unpaired nucleotides. Arabidopsis thaliana and other plants encode MutS protein homologs (MSH) conserved among other eukaryotic organisms, but also encode an extra MSH polypeptide (MSH7). In order to better understand the role of MSH7 in vivo, a full set of phenotypic parameters that covered the development of the plant from seed imbibition to flowering and seed maturation was analyzed in A. thaliana harboring two different msh7 alleles. Plants deficient in MSH7 show statistically significant faster germination rates, longer primary roots during the juvenile vegetative phase, and higher cauline leaf and axillary and lateral inflorescence numbers compared with wild type. We also quantified number, length and area of siliques and seed number per silique. Disruption of MSH7 resulted in a higher number of smaller siliques than wild type. There were no differences in seed number per silique between genotypes. These findings suggest that mutant plant growth appears to be caused by an impaired cell cycle checkpoint that allows cell division without adequate DNA repair. This increase in proliferation activity demonstrates a functional and temporal link between DNA repair and cell cycle regulation.

Primary metabolism changes triggered in soybean leaves by Fusarium tucumaniae infection.

Sudden death syndrome (SDS) of soybean can be caused by at least four distinct Fusarium species, with F. tucumaniae being the main causal agent in Argentina. The fungus is a soil-borne pathogen that is largely confined to the roots, but damage also reaches aerial part of the plant and interveinal chlorosis and necrosis, followed by premature defoliation can be observed. In this study, two genetically diverse soybean cultivars, one susceptible (NA 4613) and one partially resistant (DM 4670) to SDS infection, were inoculated with F. tucumaniae or kept uninoculated. Leaf samples at 7, 10, 14 and 25 days post-inoculation (dpi) were chosen for analysis. With the aim of detecting early markers that could potentially discriminate the cultivar response to SDS, gas chromatography-mass spectrometry (GC-MS) analyses and biochemical studies were performed. Metabolic analyses show higher levels of several amino acids in the inoculated than in the uninoculated susceptible cultivar starting at 10 dpi. Biochemical studies indicate that pigment contents and Rubisco level were reduced while class III peroxidase activity was increased in the inoculated susceptible plant at 10 dpi. Taken together, our results indicate that the pathogen induced an accumulation of amino acids, a decrease of the photosynthetic activity, and an increase of plant-specific peroxidase activity in the susceptible cultivar before differences of visible foliar symptoms between genotypes could be observed, thus suggesting that metabolic and biochemical approaches may contribute to a rapid characterization of the cultivar response to SDS.

Protecting DNA from errors and damage: an overview of DNA repair mechanisms in plants compared to mammals.

The genome integrity of all organisms is constantly threatened by replication errors and DNA damage arising from endogenous and exogenous sources. Such base pair anomalies must be accurately repaired to prevent mutagenesis and/or lethality. Thus, it is not surprising that cells have evolved multiple and partially overlapping DNA repair pathways to correct specific types of DNA errors and lesions. Great progress in unraveling these repair mechanisms at the molecular level has been made by several talented researchers, among them Tomas Lindahl, Aziz Sancar, and Paul Modrich, all three Nobel laureates in Chemistry for 2015. Much of this knowledge comes from studies performed in bacteria, yeast, and mammals and has impacted research in plant systems. Two plant features should be mentioned. Plants differ from higher eukaryotes in that they lack a reserve germline and cannot avoid environmental stresses. Therefore, plants have evolved different strategies to sustain genome fidelity through generations and continuous exposure to genotoxic stresses. These strategies include the presence of unique or multiple paralogous genes with partially overlapping DNA repair activities. Yet, in spite (or because) of these differences, plants, especially Arabidopsis thaliana, can be used as a model organism for functional studies. Some advantages of this model system are worth mentioning: short life cycle, availability of both homozygous and heterozygous lines for many genes, plant transformation techniques, tissue culture methods and reporter systems for gene expression and function studies. Here, I provide a current understanding of DNA repair genes in plants, with a special focus on A. thaliana. It is expected that this review will be a valuable resource for future functional studies in the DNA repair field, both in plants and animals.

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.

ANTI-SILENCING FUNCTION1 proteins are involved in ultraviolet-induced DNA damage repair and are cell cycle regulated by E2F transcription factors in Arabidopsis.

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 E2F corresponds to Adenovirus E2 Binding Factor. [corrected]. 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.

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 MutSß, with subsequent inability to interact with yeast downstream proteins needed to complete MMR.

Yeast mutator phenotype enforced by Arabidopsis PMS1 expression.

The DNA mismatch repair (MMR) system is a major DNA repair pathway whose function is critical for the correction of DNA biosynthetic errors. MMR is initiated by the binding of MutS proteins to mismatches and unpaired nucleotides followed by the recruitment of MutL proteins. The major MutL activity in eukaryotes is performed by MutLα, the heterocomplex of MLH1-PMS1 in yeast and plants and MLH1-PMS2 in humans. We here report the effect the expression of Arabidopsis PMS1 protein exerts on Saccharomyces cerevisiae genomic stability. A strain carrying specific microsatellite instability reporter systems was chosen for the study. The plant protein failed to complement the hypermutator phenotype of a pms1 deficient strain but increased approximately 14-fold and 2,000-fold the mutation rates of his7-2 and lys2::InsE-A 14 loci of MMR proficient strains when compared to wild-type strains, respectively. Overexpressing AtMLH1 in the AtPMS1-overproducing strain generated an increase in mutation rate comparable to that of AtPMS1 expression alone. Deletion of the C-terminal residues implicated in protein-protein interaction and including the putative endonuclease sequence of AtPMS1 completely eliminated the mutator phenotype. Taken together, these results indicate that the plant proteins affect yeast genomic stability, very possibly altering protein-protein interactions that are necessary to complete repair.

Research on plants for the understanding of diseases of nuclear and mitochondrial origin.

Different model organisms, such as Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, mouse, cultured human cell lines, among others, were used to study the mechanisms of several human diseases. Since human genes and proteins have been structurally and functionally conserved in plant organisms, the use of plants, especially Arabidopsis thaliana, as a model system to relate molecular defects to clinical disorders has recently increased. Here, we briefly review our current knowledge of human diseases of nuclear and mitochondrial origin and summarize the experimental findings of plant homologs implicated in each process.

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.

High-level production of MSH2 from Arabidopsis thaliana: a DNA mismatch repair system key subunit.

Biochemical and immunological information concerning DNA mismatch repair proteins from higher plants is currently limited, probably due to their low abundance in vivo. An initial analysis of AtMSH2 gene expression by quantitative real-time RT-PCR indicates that calli and seedlings contain 96.7 and 1.4 cDNA copies per ng RNA, respectively, confirming that this gene is predominantly expressed in rapidly dividing tissues. In order to obtain large quantities of AtMSH2, the protein was efficiently expressed in an Escherichia coli system. The expressed gene product has an in-frame N-terminal Trx-His(6)-S-tag. The fusion protein represents about 11% of the soluble protein from IPTG-induced E. coli cells. After a two-step purification procedure the final yield accounts for 0.7 mg/g cells. Digestion of this electrophoretically homogeneous recombinant protein with enterokinase results in an intact protein with only one extra amino acid introduced at the N-terminal end. Purified intact protein was used to induce polyclonal antibodies in rabbits. These antibodies cross-react with a 110-kDa protein from cauliflower inflorescences. Together, our data describe the transcript level, cloning, expression, purification, and polyclonal antibody preparation of AtMSH2. This work will surely be useful for carrying out plant mismatch repair assays in vitro and analyzing protein expression after the exposure of plants to various stresses.

PMS1 from Arabidopsis thaliana: optimization of protein overexpression in Escherichia coli.

One of the major limitations when attempting to obtain detailed biochemical, biophysical and immunological characterization of plant DNA mismatch repair proteins is their extremely low abundance in vivo under normal growth conditions. An initial analysis of PMS1 transcript level in various Arabidopsis thaliana tissues was carried out by quantitative real-time RT-PCR. For calli, flowers and seedlings, the corresponding cDNA copies per ng RNA were 66.9, 3.1 and 2.7, respectively. This suggests an important role of this gene in rapidly dividing tissues. In order to obtain a high level of PMS1 from Arabidopsis thaliana, the protein production was successfully optimized in an Escherichia coli host. The corresponding coding sequence of PMS1 was inserted into pET28a downstream a hexa-histidyl leader sequence. The pET28a-AtPMS1 plasmid was efficiently expressed in JM109(DE3)-pRIL strain probably due to the genotype features of the cells (endA1, recA1, relA1, Δ(lac-proAB), laqIqZΔM15) and the presence of extra copies of argU, ileY, and leuW tRNA genes, which encode the RIL codons. This strategy has allowed us to obtain His-tagged PMS1 at about 7% of the total soluble E. coli cell protein. The protein was purified by standard Ni(+) affinity chromatography procedures and the electrophoretically homogeneous preparation was used as an antigen for antibody generation in rabbits. This approach provides effective tools for a further reconstitution of plant mismatch repair (MMR) system in vitro and for the analysis of protein expression and distribution of AtPMS1 in various tissues after different treatments (e.g. DNA mutagens).

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.

Conformational changes of maize and wheat NADP-malic enzyme studied by quenching of protein native fluorescence.

Quenching of tryptophan fluorescence of maize and wheat NADP-malic enzyme by KI and acrylamide was studied after denaturating proteins with guanidine hydrochloride, and subjecting them to different pH values or temperatures. Protein unfolding by guanidine hydrochloride resulted in a red shift of the fluorescence spectrum, providing further support for the motion that several of the tryptophan residues evolved from an apolar to a polar environment. Protein denaturation was accompanied by an increase in the effective dynamic quenching constant values and by loss of the enzyme's activities. Thermal denaturation gave results consistent with the ones observed for chemical denaturation suggesting that a putative intermediate is involved in the denaturation process. Finally, exposure of both enzymes at various pH values allowed us to infer the number of accessible tryptophan residues in the different oligomeric conformations. The results suggest that the aggregation process seems to be different for each enzyme. Thus, as the maize enzyme associated from monomer to tetramer, one tryptophan residue would change from a polar to an apolar environment, while the association of the wheat enzyme would cause that two tryptophan residues to be excluded from quenching. Hitherto, quenching of the tryptophan fluorescence provides a good tool for studying conformational changes of proteins. The future availability of the crystal structures of plant NADP-malic enzymes will offer a good validation point for our model and the technology used.