New perspectives on the plant PARP family: Arabidopsis PARP3 is inactive, and PARP1 exhibits predominant poly (ADP-ribose) polymerase activity in response to DNA damage.

BACKGROUND: Poly (ADP-ribosyl) ation (PARylation) is an important posttranslational modification that regulates DNA repair, gene transcription, stress responses and developmental processes in multicellular organisms. Poly (ADP-ribose) polymerase (PARP) catalyzes PARylation by consecutively adding ADP-ribose moieties from NAD+ to the amino acid receptor residues on target proteins. Arabidopsis has three canonical PARP members, and two of these members, AtPARP1 and AtPARP2, have been demonstrated to be bona fide poly (ADP-ribose) polymerases and to regulate DNA repair and stress response processes. However, it remains unknown whether AtPARP3, a member that is highly expressed in seeds, has similar biochemical activity to that of AtPARP1 and AtPARP2. Additionally, although both the phylogenetic relationships and structural similarities indicate that AtPARP1 and AtPARP2 correspond to animal PARP1 and PARP2, respectively, two previous studies have indicated that AtPARP2, and not AtPARP1, accounts for most of the PARP activity in Arabidopsis, which is contrary to the knowledge that PARP1 is the predominant PARP in animals. RESULTS: In this study, we obtained both in vitro and in vivo evidence demonstrating that AtPARP3 does not act as a typical PARP in Arabidopsis. Domain swapping and point mutation assays indicated that AtPARP3 has lost NAD+-binding capability and is inactive. In addition, our results showed that AtPARP1 was responsible for most of the PARP enzymatic activity in response to the DNA damage-inducing agents zeocin and methyl methanesulfonate (MMS) and was more rapidly activated than AtPARP2, which supports that AtPARP1 remains the predominant PARP member in Arabidopsis. AtPARP1 might first become activated by binding to damaged sites, and AtPARP2 is then poly (ADP-ribosyl) ated by AtPARP1 in vivo. CONCLUSIONS: Collectively, our biochemical and genetic analysis results strongly support the notion that AtPARP3 has lost poly (ADP-ribose) polymerase activity in plants and performs different functions from those of AtPARP1 and AtPARP2. AtPARP1, instead of AtPARP2, plays the predominant role in PAR synthesis in both seeds and seedlings. These data bring new insights into our understanding of the physiological functions of plant PARP family members.

The AWPM-19 Family Protein OsPM1 Mediates Abscisic Acid Influx and Drought Response in Rice.

Abscisic acid (ABA) regulates plant stress responses and development. However, how the ABA signal is transmitted in response to stresses remains largely unclear, especially in monocots. In this study, we found that rice (Oryza sativa) OsPM1 (PLASMA MEMBRANE PROTEIN1), encoded by a gene of AWPM-19 like family, mediates ABA influx through the plasma membrane. OsPM1 is predominantly expressed in vascular tissues, guard cells, and mature embryos. Phenotypic analysis of overexpression, RNA interference (RNAi), and knockout (KO) lines showed that OsPM1 is involved in drought responses and seed germination regulation. 3H-(±)ABA transport activity and fluorescence resonance energy transfer assays both demonstrated that OsPM1 facilitates ABA uptake into cells. The physiological isomer of ABA, (+)-ABA, is the preferred substrate of OsPM1. Higher ABA accumulation and faster stomatal closure in response to ABA treatment were observed in the overexpression lines compared with the wild-type control. Many ABA-responsive genes were upregulated more in the OsPM1-overexpression lines but less in the RNAi lines compared with wild-type plants. Further investigation revealed that OsPM1 expression is regulated by the AREB/ABF family transcription factor OsbZIP46. Our results thus revealed that OsPM1 is an ABA influx carrier that plays an important role in drought responses.

Overexpressing Exogenous 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) Genes Increases Fecundity and Auxin Content of Transgenic Arabidopsis Plants.

Transgenic glyphosate-tolerant plants overproducing EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) may exhibit enhanced fitness in glyphosate-free environments. If so, introgression of transgenes overexpressing EPSPS into wild relative species may lead to increased competitiveness of crop-wild hybrids, resulting in unpredicted environmental impact. Assessing fitness effects of transgenes overexpressing EPSPS in a model plant species can help address this question, while elucidating how overproducing EPSPS affects the fitness-related traits of plants. We produced segregating T2 and T3Arabidopsis thaliana lineages with or without a transgene overexpressing EPSPS isolated from rice or Agrobacterium (CP4). For each of the three transgenes, we compared glyphosate tolerance, some fitness-related traits, and auxin (indole-3-acetic acid) content in transgene-present, transgene-absent, empty vector (EV), and parental lineages in a common-garden experiment. We detected substantially increased glyphosate tolerance in T2 plants of transgene-present lineages that overproduced EPSPS. We also documented significant increases in fecundity, which was associated with increased auxin content in T3 transgene-present lineages containing rice EPSPS genes, compared with their segregating transgene-absent lineages, EV, and parental controls. Our results from Arabidopsis with nine transgenic events provide a strong support to the hypothesis that transgenic plants overproducing EPSPS can benefit from a fecundity advantage in glyphosate-free environments. Stimulated biosynthesis of auxin, an important plant growth hormone, by overproducing EPSPS may play a role in enhanced fecundity of the transgenic Arabidopsis plants. The obtained knowledge is useful for assessing environmental impact caused by introgression of transgenes overproducing EPSPS from any GE crop into populations of its wild relatives.

Arabidopsis PARG1 is the key factor promoting cell survival among the enzymes regulating post-translational poly(ADP-ribosyl)ation.

Poly(ADP-ribosyl)ation is a reversible post-translational modification of proteins, characterized by the addition of poly(ADP-ribose) (PAR) to proteins by poly(ADP-ribose) polymerase (PARP), and removal of PAR by poly(ADP-ribose) glycohydrolase (PARG). Three PARPs and two PARGs have been found in Arabidopsis, but their respective roles are not fully understood. In this study, the functions of each PARP and PARG in DNA repair were analyzed based on their mutant phenotypes under genotoxic stresses. Double or triple mutant analysis revealed that PARP1 and PARP2, but not PARP3, play a similar but not critical role in DNA repair in Arabidopsis seedlings. PARG1 and PARG2 play an essential and a minor role, respectively under the same conditions. Mutation of PARG1 results in increased DNA damage level and enhanced cell death in plants after bleomycin treatment. PARG1 expression is induced primarily in root and shoot meristems by bleomycin and induction of PARG1 is dependent on ATM and ATR kinases. PARG1 also antagonistically modulates the DNA repair process by preventing the over-induction of DNA repair genes. Our study determined the contribution of each PARP and PARG member in DNA repair and indicated that PARG1 plays a critical role in this process.