Project description:Aluminum (Al) toxicity and phosphate (Pi) limitation are widespread chronic abiotic and mutually enhancing stresses that profoundly affect crop yield. Both stresses causes a strong inhibition of root growth due to a progressive exhaustion of the stem cell niche. Here we report on a CASEIN KINASE 2 (CK2) inhibitor identified by its capability to maintain a functional root stem cell niche under Al toxic conditions. CK2 operates through phosphorylation of the cell cycle checkpoint activator SUPPRESSOR OF GAMMA RADIATION 1 (SOG1), priming its activity under DNA damaging conditions. In addition to yielding Al tolerance, both CK2 and SOG1 inactivation result in resistance to phosphate starvation, revealing the existence of a low phosphate induced cell cycle checkpoint that depends on the DNA damage activator ATAXIA-TELANGIECTASIA MUTATED (ATM). Overall, our data reveal an important physiological role for the plant DNA damage response pathway under agricultural limiting growth conditions and opening new avenues to cope with the problem of Pi limitation. SOG1 can be phosphorylated by both ATM and ATR (Sjogren et al., 2015; Yoshiyama et al., 2017). ATM phosphorylation of SOG1 can occur at five different S/TQ sites upon treatment with double-stand break inducing compounds such as zeocin. This phosphorylation is important for its activity, as demonstrated by the inability of a phosphor-mutant allele to complement the sog1 mutant. In addition to the ATM/ATR-dependent phosphorylation sites, 11 putative CK2 phosphorylation sites [T/SXXE/D, with S or T serving as the phospho-acceptor site (Meggio and Pinna, 2003)] are found within the full length SOG1 proteins. Among these, four sites are conserved among orthologous proteins, stressing their potential importance to protein function. Of these, three cluster at the extreme C-terminus of SOG1, which also is also the location of the ATM/ATR phosphorylation sites. To test putative phosphorylation of any of these sites, TAP-tagged SOG1 protein was extracted and purified from cell cultures and subjected to phosphorylation analysis.

Project description:It has been strongly argued that plant cells should have a means of sensing sugars at the cell surface, so that extracellular and intracellular sugars can be sensed separately and their metabolism coordinated (Lalonde et al., Plant Cell, 11, 707-26, 2000). There is good evidence for an intracellular hexokinase-dependent pathway of hexose sensing in plants, but very little evidence for a hexokinase-independent signalling pathway, such as that provided by SNF3 or RGT2 in yeast. Many papers on sugar sensing in plants cite work from two laboratories as evidence for hexokinase-independent hexose signalling in plants. The first is that in which cell-wall invertase and sucrose synthase genes were induced by treatment of a Chenopodium suspension culture with 30 mM 6-Deoxyglucose (6DOG) for 24 h (Roitsch et al., Plant Physiol 108, 285-294, 1995; Godt et al., J. Plant Physiol 146, 231-238, 1995). The second is that in which a patatin transgene in Arabidopsis was shown to be weakly induced by growth over several days on a mixture of 30 mM glucose plus 30 mM 3-O-methylglucose (3OMG), but strongly induced by growth on 30 mM Glc plus 90 mM 3OMG (Martin et al., Plant J, 11, 53-62, 1997). We are not aware of any examples of Arabidopsis genes which respond to 6DOG or 3OMG yet this is an area of wide significance. Identification of such a gene would help to establish if a hexokinase-independent signalling system operates in plants, and would provide a basis for establishment of a genetic screen for mutants, using the gene promoter linked to a reporter such as luciferase. The aim of this proposal is to discover any genes which are either activated or repressed by glucose AND by 3OMG and/or 6DOG, but not by mannitol (an osmotic control). The use of both 3OMG and 6DOG will help to identify non-specific effects of either. All substrates will first be analysed by HPLC to confirm that they are pure. Arabidopsis Col-0 seedlings will be grown in vitro for 7 days in the absence of sugars, then treated with 30 mM glucose or glucose analogue for 8 h (these conditions are based on concentrations and time courses of Roitsch et al.). RNA will then be isolated from multiple independent plates to minimise biological variation. Experimenter name = Dorthe Villadsen Experimenter phone = 0131 650 5318 Experimenter fax = 0131 650 5392 Experimenter address = Institute of Cell and Molecular Biology Experimenter address = University of Edinburgh Experimenter address = The King_s Buildings Experimenter address = Mayfield Road Experimenter address = Edinburgh Experimenter zip/postal_code = EH9 3JH Experimenter country = UK Keywords: growth_condition_design

Project description:It has been strongly argued that plant cells should have a means of sensing sugars at the cell surface, so that extracellular and intracellular sugars can be sensed separately and their metabolism coordinated (Lalonde et al., Plant Cell, 11, 707-26, 2000). There is good evidence for an intracellular hexokinase-dependent pathway of hexose sensing in plants, but very little evidence for a hexokinase-independent signalling pathway, such as that provided by SNF3 or RGT2 in yeast. Many papers on sugar sensing in plants cite work from two laboratories as evidence for hexokinase-independent hexose signalling in plants. The first is that in which cell-wall invertase and sucrose synthase genes were induced by treatment of a Chenopodium suspension culture with 30 mM 6-Deoxyglucose (6DOG) for 24 h (Roitsch et al., Plant Physiol 108, 285-294, 1995; Godt et al., J. Plant Physiol 146, 231-238, 1995). The second is that in which a patatin transgene in Arabidopsis was shown to be weakly induced by growth over several days on a mixture of 30 mM glucose plus 30 mM 3-O-methylglucose (3OMG), but strongly induced by growth on 30 mM Glc plus 90 mM 3OMG (Martin et al., Plant J, 11, 53-62, 1997). We are not aware of any examples of Arabidopsis genes which respond to 6DOG or 3OMG yet this is an area of wide significance. Identification of such a gene would help to establish if a hexokinase-independent signalling system operates in plants, and would provide a basis for establishment of a genetic screen for mutants, using the gene promoter linked to a reporter such as luciferase. The aim of this proposal is to discover any genes which are either activated or repressed by glucose AND by 3OMG and/or 6DOG, but not by mannitol (an osmotic control). The use of both 3OMG and 6DOG will help to identify non-specific effects of either. All substrates will first be analysed by HPLC to confirm that they are pure. Arabidopsis Col-0 seedlings will be grown in vitro for 7 days in the absence of sugars, then treated with 30 mM glucose or glucose analogue for 8 h (these conditions are based on concentrations and time courses of Roitsch et al.). RNA will then be isolated from multiple independent plates to minimise biological variation.

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Genome-wide identification of SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1)-target genes in Arabidopsis

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