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Analysis of knockout mutants reveals non-redundant functions of poly(ADP-ribose)polymerase isoforms in Arabidopsis.

Pham PA, Wahl V, Tohge T, de Souza LR, Zhang Y, Do PT, Olas JJ, Stitt M, Araújo WL, Fernie AR - Plant Mol. Biol. (2015)

Bottom Line: We next isolated and characterized insertional knockout mutants of all three isoforms confirming a complete knockout in the full length transcript levels of the target genes as well as a reduced total leaf NAD hydrolase activity in the two isoforms (PARP1, PARP2) that are highly expressed in leaves.Physiological evaluation of the mutant lines revealed that they displayed distinctive metabolic and root growth characteristics albeit unaltered leaf morphology under optimal growth conditions.We therefore conclude that the PARP isoforms play non-redundant non-nuclear metabolic roles and that their function is highly important in rapidly growing tissues such as the shoot apical meristem, roots and seeds.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476, Potsdam-Golm, Germany.

ABSTRACT
The enzyme poly(ADP-ribose)polymerase (PARP) has a dual function being involved both in the poly(ADP-ribosyl)ation and being a constituent of the NAD(+) salvage pathway. To date most studies, both in plant and non-plant systems, have focused on the signaling role of PARP in poly(ADP-ribosyl)ation rather than any role that can be ascribed to its metabolic function. In order to address this question we here used a combination of expression, transcript and protein localization studies of all three PARP isoforms of Arabidopsis alongside physiological analysis of the corresponding mutants. Our analyses indicated that whilst all isoforms of PARP were localized to the nucleus they are also present in non-nuclear locations with parp1 and parp3 also localised in the cytosol, and parp2 also present in the mitochondria. We next isolated and characterized insertional knockout mutants of all three isoforms confirming a complete knockout in the full length transcript levels of the target genes as well as a reduced total leaf NAD hydrolase activity in the two isoforms (PARP1, PARP2) that are highly expressed in leaves. Physiological evaluation of the mutant lines revealed that they displayed distinctive metabolic and root growth characteristics albeit unaltered leaf morphology under optimal growth conditions. We therefore conclude that the PARP isoforms play non-redundant non-nuclear metabolic roles and that their function is highly important in rapidly growing tissues such as the shoot apical meristem, roots and seeds.

No MeSH data available.


PARP enzymatic assay calibration curve. Leaf nuclei of Col-0 grown in long day (16 h photoperiod) were isolated. Filled black circles represent enzymatic assay performed with addition of 1 mM NAD+ as substrate with different incubation time (20-40-80-120-180 min). Filled red circles represent data of enzymatic assay performed with addition of different substrate concentration (0.5-1-3 mM NAD+) and 120 min incubation time
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Fig6: PARP enzymatic assay calibration curve. Leaf nuclei of Col-0 grown in long day (16 h photoperiod) were isolated. Filled black circles represent enzymatic assay performed with addition of 1 mM NAD+ as substrate with different incubation time (20-40-80-120-180 min). Filled red circles represent data of enzymatic assay performed with addition of different substrate concentration (0.5-1-3 mM NAD+) and 120 min incubation time

Mentions: As mentioned above, PARP uses NAD+ as substrate in the salvage pathway to produce NaM via poly(ADP-ribosyl)ation. In order to investigate whether the decreases in PARP transcript resulted in similar changes at the enzyme activity level, an assay was established to determine the production of NaM in whole cell extracts as well as nuclei preparations from leaf tissue. This approach was taken given the difficulty in detecting poly(ADP-ribose) and as a means to discriminate PARP activity from that of the parallel reaction catalyzed by sirtuin (König et al. 2014) and the similar reaction catalyzed by nudix enyzmes (Hashida et al. 2009), although it is important to note that it may not discriminate PARP from other potential NAD+ hydrolase activities. Thus we are only able to quantify the total NAD+ hydrolase activity by this approach. For this purpose fresh Arabidopsis rosette leaf tissue was ground and incubated in a simple isolation buffer as described previously by (Folta and Kaufman 2006) prior to filtering through two layers of miracloth. To isolate nuclei, the filtered suspension was then lysed and centrifuged to enrich for nuclei. To remove metabolites or peptides, which could interfere with the reaction, a desalting step was carried out. The enzymatic assay was then performed with a 1 mM NAD+-containing enzymatic reaction buffer for 60 min at room temperature and stopped by addition of ice-cold trichloroacetic acid. The NaM produced in the end-point assay was subsequently extracted and measured by gas chromatography-mass spectrometry (GC–MS) as detailed in the “Materials and methods” section. Given that this was an assay novel to us, before analyzing the mutant lines we verified that it was linear with respect to both time and protein concentration (data not shown). The cellular NAD+ hydrolysis activity of wild type Arabidopsis observed (Fig. 6) was similar to those previously reported for PARP in mammals and maize (Grube and Bürkle 1992; Tian et al. 1999). A reduced activity was observed for the parp1 and parp2 mutants in both extracts of whole leaf extracts and nuclei preparations, although the reduction was statistically significant only for the nuclei (Fig. 7). Expectedly, the activity in parp3 mutant was at wild type level, since PARP3 is not highly expressed in vegetative tissue, however importantly the enhanced expression of PARP1 in this line did not result in an increased overall PARP activity.Fig. 6


Analysis of knockout mutants reveals non-redundant functions of poly(ADP-ribose)polymerase isoforms in Arabidopsis.

Pham PA, Wahl V, Tohge T, de Souza LR, Zhang Y, Do PT, Olas JJ, Stitt M, Araújo WL, Fernie AR - Plant Mol. Biol. (2015)

PARP enzymatic assay calibration curve. Leaf nuclei of Col-0 grown in long day (16 h photoperiod) were isolated. Filled black circles represent enzymatic assay performed with addition of 1 mM NAD+ as substrate with different incubation time (20-40-80-120-180 min). Filled red circles represent data of enzymatic assay performed with addition of different substrate concentration (0.5-1-3 mM NAD+) and 120 min incubation time
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4631723&req=5

Fig6: PARP enzymatic assay calibration curve. Leaf nuclei of Col-0 grown in long day (16 h photoperiod) were isolated. Filled black circles represent enzymatic assay performed with addition of 1 mM NAD+ as substrate with different incubation time (20-40-80-120-180 min). Filled red circles represent data of enzymatic assay performed with addition of different substrate concentration (0.5-1-3 mM NAD+) and 120 min incubation time
Mentions: As mentioned above, PARP uses NAD+ as substrate in the salvage pathway to produce NaM via poly(ADP-ribosyl)ation. In order to investigate whether the decreases in PARP transcript resulted in similar changes at the enzyme activity level, an assay was established to determine the production of NaM in whole cell extracts as well as nuclei preparations from leaf tissue. This approach was taken given the difficulty in detecting poly(ADP-ribose) and as a means to discriminate PARP activity from that of the parallel reaction catalyzed by sirtuin (König et al. 2014) and the similar reaction catalyzed by nudix enyzmes (Hashida et al. 2009), although it is important to note that it may not discriminate PARP from other potential NAD+ hydrolase activities. Thus we are only able to quantify the total NAD+ hydrolase activity by this approach. For this purpose fresh Arabidopsis rosette leaf tissue was ground and incubated in a simple isolation buffer as described previously by (Folta and Kaufman 2006) prior to filtering through two layers of miracloth. To isolate nuclei, the filtered suspension was then lysed and centrifuged to enrich for nuclei. To remove metabolites or peptides, which could interfere with the reaction, a desalting step was carried out. The enzymatic assay was then performed with a 1 mM NAD+-containing enzymatic reaction buffer for 60 min at room temperature and stopped by addition of ice-cold trichloroacetic acid. The NaM produced in the end-point assay was subsequently extracted and measured by gas chromatography-mass spectrometry (GC–MS) as detailed in the “Materials and methods” section. Given that this was an assay novel to us, before analyzing the mutant lines we verified that it was linear with respect to both time and protein concentration (data not shown). The cellular NAD+ hydrolysis activity of wild type Arabidopsis observed (Fig. 6) was similar to those previously reported for PARP in mammals and maize (Grube and Bürkle 1992; Tian et al. 1999). A reduced activity was observed for the parp1 and parp2 mutants in both extracts of whole leaf extracts and nuclei preparations, although the reduction was statistically significant only for the nuclei (Fig. 7). Expectedly, the activity in parp3 mutant was at wild type level, since PARP3 is not highly expressed in vegetative tissue, however importantly the enhanced expression of PARP1 in this line did not result in an increased overall PARP activity.Fig. 6

Bottom Line: We next isolated and characterized insertional knockout mutants of all three isoforms confirming a complete knockout in the full length transcript levels of the target genes as well as a reduced total leaf NAD hydrolase activity in the two isoforms (PARP1, PARP2) that are highly expressed in leaves.Physiological evaluation of the mutant lines revealed that they displayed distinctive metabolic and root growth characteristics albeit unaltered leaf morphology under optimal growth conditions.We therefore conclude that the PARP isoforms play non-redundant non-nuclear metabolic roles and that their function is highly important in rapidly growing tissues such as the shoot apical meristem, roots and seeds.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476, Potsdam-Golm, Germany.

ABSTRACT
The enzyme poly(ADP-ribose)polymerase (PARP) has a dual function being involved both in the poly(ADP-ribosyl)ation and being a constituent of the NAD(+) salvage pathway. To date most studies, both in plant and non-plant systems, have focused on the signaling role of PARP in poly(ADP-ribosyl)ation rather than any role that can be ascribed to its metabolic function. In order to address this question we here used a combination of expression, transcript and protein localization studies of all three PARP isoforms of Arabidopsis alongside physiological analysis of the corresponding mutants. Our analyses indicated that whilst all isoforms of PARP were localized to the nucleus they are also present in non-nuclear locations with parp1 and parp3 also localised in the cytosol, and parp2 also present in the mitochondria. We next isolated and characterized insertional knockout mutants of all three isoforms confirming a complete knockout in the full length transcript levels of the target genes as well as a reduced total leaf NAD hydrolase activity in the two isoforms (PARP1, PARP2) that are highly expressed in leaves. Physiological evaluation of the mutant lines revealed that they displayed distinctive metabolic and root growth characteristics albeit unaltered leaf morphology under optimal growth conditions. We therefore conclude that the PARP isoforms play non-redundant non-nuclear metabolic roles and that their function is highly important in rapidly growing tissues such as the shoot apical meristem, roots and seeds.

No MeSH data available.