Chemical PARP Inhibition Enhances Growth of Arabidopsis and Reduces Anthocyanin Accumulation and the Activation of Stress Protective Mechanisms

Poly-ADP-ribose polymerase (PARP) post-translationally modifies proteins through the addition of ADP-ribose polymers, yet its role in modulating plant development and stress responses is only poorly understood. The experiments presented here address some of the gaps in our understanding of its role in stress tolerance and thereby provide new insights into tolerance mechanisms and growth. Using a combination of chemical and genetic approaches, this study characterized phenotypes associated with PARP inhibition at the physiological level. Molecular analyses including gene expression analysis, measurement of primary metabolites and redox metabolites were used to understand the underlying processes. The analysis revealed that PARP inhibition represses anthocyanin and ascorbate accumulation under stress conditions. The reduction in defense is correlated with enhanced biomass production. Even in unstressed conditions protective genes and molecules are repressed by PARP inhibition. The reduced anthocyanin production was shown to be based on the repression of transcription of key regulatory and biosynthesis genes. PARP is a key factor for understanding growth and stress responses of plants. PARP inhibition allows plants to reduce protection such as anthocyanin, ascorbate or Non-Photochemical-Quenching whilst maintaining high energy levels likely enabling the observed enhancement of biomass production under stress, opening interesting perspectives for increasing crop productivity

Adapting crops to climate Change

The growing world population forces us to invest in increasing research into plants that will grow on less than optimal land while there is and increasing need for alternative energy from biofeedstocks. To be successful, businesses need to cooperate with public sector scientists at all levels

Mini-scale method for nuclear run-on transcription assay in plants

We present a mini-scale method for nuclear run-on transcription assay. In our method, all the centrifuge steps can be carried out by using micro-tubes for short time (5 min each) throughout the process, including isolation of transcriptionally active nuclei and purification of labeled RNA after synthesis of RNA in isolated nuclei. The assay can be performed using a small amount of plant tissue, which enables analysis of developmental changes in transcriptional status of given genes in a single individual plant. Successful results were obtained using the tissues of flower and leaf of petunia and embryo of pea, suggesting that the method is potentially applicable to a variety of plant tissues

Developmentally and transgene regulated nuclear processing of primary transcripts of chalcone synthase A in petunia

The introduction of chalcone synthase A transgenes into petunia plants can result in degradation of chalcone synthase A RNAs and loss of chalcone synthase, a process called cosuppression or post-transcriptional gene silencing. Here we show that the RNA degradation is associated with changes in premRNA processing, i.e. loss of tissue specificity in transcript cleavage patterns, accumulation of unspliced molecules, and use of template-specific secondary poly(A) sites. These changes can also be observed at a lower level in leaves but not flowers of nontransgenic petunias. Based on this, a model is presented of how transgenes may disturb the carefully evolved, developmentally controlled post-transcriptional regulation of chalcone synthase gene expression by influencing the survival rate of the endogenous and their own mRNA

Epigenetic inactivation of chalcone synthase-A transgene transcription in petunia leads to a reversion of the post-transcriptional gene silencing phenotype

Petunia plants that exhibit a white-flowering phenotype as a consequence of chalcone synthase transgene-induced silencing occasionally give rise to revertant branches that produce flowers with wild-type pigmentation. Transcription run-on assays confirmed that the production of white flowers is caused by post-transcriptional gene silencing (PTGS), and indicated that transgene transcription is repressed in the revertant plants, providing evidence that induction of PTGS depends on the transcription rate. Transcriptional repression of the transgene was associated with cytosine methylation at CpG, CpNpG and CpNpN sites, and the expression was restored by treatment with either 5-azacytidine or trichostatin A. These results demonstrate that epigenetic changes occurred in the PTGS line, and these changes interfere with the initiation of transgene transcription, leading to a reversion of the PTGS phenotype

Dose response of plants to chemical PARP inhibition.

<p><b>A</b>, <i>Arabidopsis thaliana</i> (Col-0) and PARPsig::RNAi (sigPARP) seedlings were grown for 21 days at 80–100 µE, 22°C on MS media containing different concentrations (mM) of the PARP inhibitor 3-Methoxy-benzamide (3 MB). The average individual fresh weight was determined by weighing 32 pooled seedlings from each plate, with four replicates (plates) in each experiment, repeated in two independent experiments (n = 8). Asterisks indicate significant (P<0.05) difference between Col-0 and PARPsig::RNAi seedlings grown without and those treated with PARP inhibitor. B, Arabidopsis plants were grown at 80–100 µE, 22°C on half MS medium and subjected to short- or long-term stress in the presence (+3 MB) or absence (−3 MB) of 0.2 mM of the PARP inhibitor 3 MB in the medium. For the short-term stress, 14 day old plants were transferred to either 450 µE (high-light) for two days and harvested at day 19 after three days of recovery or to 40°C (heat) for 6 h and harvested after seven days of recovery (day 21). For the long-term stress, plants were grown for 21 days either in control, 0.1 µM Paraquat (oxidative) or 75 mM NaCl (salt) stress conditions. Fresh weight was determined by weighing a pool of 32 seedlings from each plate, with four replicates (plates) in each experiment repeated in two independent experiments (n = 8). Asterisks indicate treatments which are significant different (P<0.05) compared to the Col-0 grown on 0 mM 3 MB.</p

Chemical PARP inhibition changes cellular redox profiles.

<p>Arabidopsis Col-0 seedlings were grown for 14 days at 80–100 µE, 22°C on MS media with (+3 MB) or without (−3 MB) the PARP inhibitor 3-Methoxy-benzamide (3 MB) and were subjected to three different treatments: control, oxidative stress (0.1 µM Paraquat) or sucrose stress (150 mM sucrose). Shown are (A) the NAD+ content, (B) the total ascorbate content, (C) the reduction level of the ascorbate, (D) the total glutathione and (E) the reduction of the total glutathione. Data are combined from three independent experiments with 2 replicates in each experiment (n = 6). Asterisks indicate significant difference (P<0.05) compared to Col-0 grown in the same condition without 3 MB.</p