Poly(ADP-ribose)polymerase activity controls plant growth by promoting leaf cell number

A changing global environment, rising population and increasing demand for biofuels are challenging agriculture and creating a need for technologies to increase biomass production. Here we demonstrate that the inhibition of poly (ADPribose) polymerase activity is a promising technology to achieve this under non-stress conditions. Furthermore, we investigate the basis of this growth enhancement via leaf series and kinematic cell analysis as well as single leaf transcriptomics and plant metabolomics under non-stress conditions. These data indicate a regulatory function of PARP within cell growth and potentially development. PARP inhibition enhances growth of Arabidopsis thaliana by enhancing the cell number. Time course single leaf transcriptomics shows that PARP inhibition regulates a small subset of genes which are related to growth promotion, cell cycle and the control of metabolism. This is supported by metabolite analysis showing overall changes in primary and particularly secondary metabolism. Taken together the results indicate a versatile function of PARP beyond its previously reported roles in controlling plant stress tolerance and thus can be a useful target for enhancing biomass production

rosettR: protocol and software for seedling area and growth analysis

Growth is an important parameter to consider when studying the impact of treatments or mutations on plant physiology. Leaf area and growth rates can be estimated efficiently from images of plants, but the experiment setup, image analysis, and statistical evaluation can be laborious, often requiring substantial manual effort and programming skills. Here we present rosettR, a non-destructive and high-throughput phenotyping protocol for the measurement of total rosette area of seedlings grown in plates in sterile conditions. We demonstrate that our protocol can be used to accurately detect growth differences among different genotypes and in response to light regimes and osmotic stress. rosettR is implemented as a package for the statistical computing software R and provides easy to use functions to design an experiment, analyze the images, and generate reports on quality control as well as a final comparison across genotypes and applied treatments. Experiment procedures are included as part of the package documentation. Using rosettR it is straight-forward to perform accurate, reproducible measurements of rosette area and relative growth rate with high-throughput using inexpensive equipment. Suitable applications include screening mutant populations for growth phenotypes visible at early growth stages and profiling different genotypes in a wide variety of treatments

PARP inhibition and its effect on the phenylpropanoid pathway.

<p>Arabidopsis shoots were harvested at day7 after transfer. Per replicate 10–12 seedlings were harvested, with 5 biological replicates in each of the three independent experiments (n = 15). The metabolite content relative to the control (no 3MB) is shown and when changed significantly the relative content is underlined. Only metabolites with a clear annotation and positioning within the pathway are shown.</p

PARP inhibition induced cell number increase.

<p>Arabidopsis leaf two was analyzed after two days of transferring the seedlings (day2) or after seven days (day7) either to treatment or control conditions. (A) Cell number per mm², (B) cell size in µm and (C) the calculated cell number per leaf is presented. Seedlings were previous grown for seven days in control conditions. In each experiment 4–6 leafs were analyzed and the experiment repeated three times independently (n = 12–18). Significant differences (P<0.05) between treated and untreated plants is indicated by an asterisk.</p

PARP inhibition affects redox metabolite concentrations.

<p>Arabidopsis shoots were harvested at day7 after transfer. Per replicate 10–12 seedlings were harvested, with 5 biological replicates in each of the three independent experiments (n = 15) and analyzed using LC-MS. The relevant data of the redox metabolites was extracted from the LC-MS data set.</p

PARP inhibition enhances growth and biomass in control conditions.

<p>Hydroponically grown plants were analyzed in respect to leaf area and biomass. 18 plants per experiment and treatment were analyzed, repeated in four experiments and the average leaf area of plants at day 26 (A) or shoot biomass (B), root biomass (C) and the shoot to root ration (D) of plants harvested day 30 is shown. Significant differences (P<0.05) between treated (0.2 mM 3MB) and untreated (125 µl DMSO) plants is indicated by an asterisk.</p

Ploidy analysis of leaf two.

<p>Arabidopsis leaf two was analyzed at the indicated time points from plants subjected to PARP inhibition or control conditions. (A) the calculated EI is shown, EI represents the number of endo cycles undergone by a typical nuclei (EI  =  1*4N+2*8N+3*16N), in (B–D) percentage of 2N, 4N and 8N nuclei is presented. 10–12 leaf two were pooled for a replicate, four biological replicates were analyzed in each of the two independent experiments (n = 8).</p

PARP activity and effects on primary metabolism.

<p>The metabolite profile of Arabidopsis shoots was analyzed by GC-MS at the indicated times following transfer of the seedlings after 7 days growth on control media to either control or PARP inhibitor containing media. Plants were grown in parallel to those for the physiological and microarray analysis. Five individual samples, each a pool of 10–12 plants, were harvested and analyzed in three independent experiments (n = 15). Amino, organic and fatty acids with significant changes in their relative abundance are shown. Metabolite content relative to the (−3MB), with red and blue indicating accumulation and depletion, are shown.</p

PARP inhibition and its effect on guard cell development.

<p>Arabidopsis leaf two was analyzed after two days of transferring the seedlings (day2) or after seven days (day7) either to treatment or control conditions. (A) The stomata index (SI) is shown, calculated by number of stomata divided by number of cells, (B) the number of stomata per mm² and (C) the calculated total number of stomata per leaf is presented. In each experiment 4–6 leafs were analyzed and the experiment repeated three times independently (n = 12–18). Significant differences (P<0.05) between treated and untreated plants is indicated by an asterisk.</p

MAPMAN metabolic overview of transcriptional changes at day7.

<p>Shown are the overall gene expression changes related to the metabolism day7 between 3MB treated leaves 2 and controls. The Wilcoxon rank sum, with Benjamini-Hochberg correction, is shown below.</p