Final Report Summary - ROXNP (Redox Regulation of Nuclear Proteins)
The tripetide thiol glutahione (gamma-glutamyl cysteinyl glycine; GSH) fulfils many important functions in plants, including antioxidant defence and redox signalling. The results obtained in this project concerning the characterisation of the functions of GSH in the model plant Arabidopsis thaliana lead to the following conclusions:
1. GSH depletion significantly increases in the redox states of the nucleus and cytosol, and causes arrest of the cell cycle in roots but not shoots, with accompanying transcript changes linked to altered hormone responses but not oxidative stress.
This conclusion is based on the following findings:
• Severe GSH depletion specifically inhibited root meristem development, while low root GSH levels decreased lateral root densities.
• The redox potential of the nucleus and cytosol of Arabidopsis thaliana roots determined using roGFP probes was between -300 to -320mV.
• Growth in the presence of the GSH synthesis inhibitor buthionine sulfoximine (BSO) increased the nuclear and cytosolic redox potentials to approximately 260mV.
• GSH-responsive genes including transcription factors (SPATULA, MYB15, MYB75), proteins involved in cell division, redox regulation (glutaredoxinS17, thioredoxins, ACHT5 and TH8) and auxin signalling (HECTATE1), were identified in the GSH-deficient root meristemless 1-1 (rml1-1) mutant, and in other GSH synthesis mutants (rax1-1, cad2-1, pad2-1) as well as in the wild type following the addition of BSO.
• Inhibition of auxin transport had no effect on organ glutathione levels but exogenous auxin decreased the root glutathione pool.
2. Glutathione has important functions in the nucleus
• The roGFP probes detected GSH in the nucleus of root cells.
• The specific fluorescent probe 5-chloromethylfluorescein diacetate (CMFDA) was also used to estimate the intracellular distribution of GSH between the nucleus and cytosol. The CMFDA stain was visible in the cytosol and nucleus of root cells but migrated within 30 minutes to the vacuole.
• Hydroxyurea was used to arrest the cell cycle at the G1/S transition.
• Nuclei with intact nuclear envelopes were purfied using the INTACT system. However, the yield of intact nuclei was very low. Alternative methods were therefore developed to purify specific nuclear proteins, particicularly histones and PARP. The analysis of redox modification of plant histones (carbonylation and poly-ADP ribosylation) was optimized in collaboration with the group of Federico Pallardo (University of Valencia, Spain). These techniques are currently being used to characterise carbonylation and poly-ADP ribosylation of histone proteins during the cell cycle.
3. Glutathione recruitment into the nucleus may involve Bcl-2 associated athanogene (BAG) proteins and the ALADIN protein
The roles of Bcl-2 associated athanogene (BAG) proteins and the nuclear pore complex (NPC) protein ALADIN in transporting GSH into the nucleus were characterised in Arabidopsis.
• Growth rates, tissue glutathione contents and the subcellular distribution of GSH were similar in the shoots and roots of the single bag mutants and the wild type plants.
• To invesigate further, the single mutants were crossed and double and triple mutants for the BAG genes were isolated.
• Growth rates, tissue glutathione contents and the subcellular distribution of GSH were similar in the shoots and roots of the double mutants.
• The bag1bag2bag3, triple mutants had an increased number of lateral roots relative to the wild-type.
• The bag1bag2bag3, accumulated less GSH in the roots and leaves than the wild-type.
• The aladin mutant shows slow growth of shoots and roots. However, GSH, ascorbate and pyridine nucleotide (NAD(P)(H)) levels were similar in the mutant and wild-type shoots and roots.
• The growth of the aladin roots was increased by GSH treatment.
• Root growth in the aladin mutants was more sensitive to GSH depletion induced by BSO than the wild-type.
• Photosynthesis was impaired in the aladin mutants relative to the wild-type.
1. GSH depletion significantly increases in the redox states of the nucleus and cytosol, and causes arrest of the cell cycle in roots but not shoots, with accompanying transcript changes linked to altered hormone responses but not oxidative stress.
This conclusion is based on the following findings:
• Severe GSH depletion specifically inhibited root meristem development, while low root GSH levels decreased lateral root densities.
• The redox potential of the nucleus and cytosol of Arabidopsis thaliana roots determined using roGFP probes was between -300 to -320mV.
• Growth in the presence of the GSH synthesis inhibitor buthionine sulfoximine (BSO) increased the nuclear and cytosolic redox potentials to approximately 260mV.
• GSH-responsive genes including transcription factors (SPATULA, MYB15, MYB75), proteins involved in cell division, redox regulation (glutaredoxinS17, thioredoxins, ACHT5 and TH8) and auxin signalling (HECTATE1), were identified in the GSH-deficient root meristemless 1-1 (rml1-1) mutant, and in other GSH synthesis mutants (rax1-1, cad2-1, pad2-1) as well as in the wild type following the addition of BSO.
• Inhibition of auxin transport had no effect on organ glutathione levels but exogenous auxin decreased the root glutathione pool.
2. Glutathione has important functions in the nucleus
• The roGFP probes detected GSH in the nucleus of root cells.
• The specific fluorescent probe 5-chloromethylfluorescein diacetate (CMFDA) was also used to estimate the intracellular distribution of GSH between the nucleus and cytosol. The CMFDA stain was visible in the cytosol and nucleus of root cells but migrated within 30 minutes to the vacuole.
• Hydroxyurea was used to arrest the cell cycle at the G1/S transition.
• Nuclei with intact nuclear envelopes were purfied using the INTACT system. However, the yield of intact nuclei was very low. Alternative methods were therefore developed to purify specific nuclear proteins, particicularly histones and PARP. The analysis of redox modification of plant histones (carbonylation and poly-ADP ribosylation) was optimized in collaboration with the group of Federico Pallardo (University of Valencia, Spain). These techniques are currently being used to characterise carbonylation and poly-ADP ribosylation of histone proteins during the cell cycle.
3. Glutathione recruitment into the nucleus may involve Bcl-2 associated athanogene (BAG) proteins and the ALADIN protein
The roles of Bcl-2 associated athanogene (BAG) proteins and the nuclear pore complex (NPC) protein ALADIN in transporting GSH into the nucleus were characterised in Arabidopsis.
• Growth rates, tissue glutathione contents and the subcellular distribution of GSH were similar in the shoots and roots of the single bag mutants and the wild type plants.
• To invesigate further, the single mutants were crossed and double and triple mutants for the BAG genes were isolated.
• Growth rates, tissue glutathione contents and the subcellular distribution of GSH were similar in the shoots and roots of the double mutants.
• The bag1bag2bag3, triple mutants had an increased number of lateral roots relative to the wild-type.
• The bag1bag2bag3, accumulated less GSH in the roots and leaves than the wild-type.
• The aladin mutant shows slow growth of shoots and roots. However, GSH, ascorbate and pyridine nucleotide (NAD(P)(H)) levels were similar in the mutant and wild-type shoots and roots.
• The growth of the aladin roots was increased by GSH treatment.
• Root growth in the aladin mutants was more sensitive to GSH depletion induced by BSO than the wild-type.
• Photosynthesis was impaired in the aladin mutants relative to the wild-type.